US20050241333A1

US20050241333A1 - Rate-controlled freezer and cooling methods thereof

Info

Publication number: US20050241333A1
Application number: US11/004,290
Authority: US
Inventors: Robert Hamilton
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2003-12-03
Filing date: 2004-12-03
Publication date: 2005-11-03

Abstract

The disclosure provides a freezer system including a temperature and cooling-rate controlled freezer having a rack system having a tray adapted for freezing and preserving temperature-sensitive items. The disclosure also provides methods for cooling and preserving items in the freezer system.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a United States non-provisional application and is related to and claims priority to co-pending U.S. provisional application Ser. No. 60/527,131, filed Dec. 3, 2003, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present disclosure generally relates to cooling devices, such as low-temperature freezers, which utilize a directly injected refrigerant, such as liquid nitrogen, to cool various products, such as temperature sensitive biological samples.
One common cryo-preservation method of biological materials involves rate-controlled freezing to minimize ice-crystal formation and to prevent osmotic effects. During cooling, ice can form at different rates depending upon, for example, the nature of the sample, such as cell permeability, and the sample's constituents and its surrounding matrix. During slow cooling, for example of biological cells, freezing typically preferentially occurs external to the cell before intracellular ice begins to form. As external ice forms, liquid water is depleted from the extra-cellular environment and an osmotic imbalance can occur across the cell membrane or cell wall, which imbalance may cause intracellular water to migrate out of the cell and lead to solute concentration within the cell. In contrast, rapid cooling minimizes osmotic effects, such as solute concentration, since ice tends to form uniformly extra-cellularly and intra-cellularly. However, rapid cooling typically produces more intracellular ice, which may cause unacceptable and irreversible damage to the cells.
U.S. Pat. No. 6,044,648, discloses a cooling device for rapidly cooling items, such as biological samples. The cooling device includes an enclosure having a chamber and an air circulation path at least partially within the chamber. A fan is operatively connected to the chamber for circulating air within the air circulation path and a perforated tube is disposed within the air circulation path for receiving a liquid refrigerant under pressure and distributing refrigerant in gaseous form to the air circulation path. Preferably the perforated tube is formed as a coil having multiple revolutions and either an axial blade fan or a centrifugal fan is received generally within the coils.
U.S. Pat. No. 5,632,388, discloses a test tube rack assembly including a test tube rack pivotally connected to a base having end support structure for allowing pivoting motion of the test tube rack with respect to a stationary base about a horizontal axis.
Controlled cooling or controlled freezing devices are available commercially, for example, the ThermoForma rate-controller freezer from Cryomed of Forma Scientific, Inc., the assignee of the above mentioned U.S. Pat. Nos. 6,044,648, and 5,632,388. However, the ThermoForma freezer and other commercially available rate-controlled cooling devices experience problems which include, for example, non-uniform cooling of samples within the device, such as, considerable relative-temperature variability among samples; considerable relative-temperature variability between the freezer's chamber and samples and within individual samples; and considerable cooling-rate variability among samples. These problems are exacerbated for large total volume sample loads.
Thus, there is a need for a freezer system, components thereof, and low-temperature preservation methods of the system or components, which are capable of providing uniform temperatures and constant rate-controlled cooling.

SUMMARY OF THE INVENTION

We have discovered a freezer system and preservation methods thereof, which provide uniform temperatures and constant rate-controlled cooling of, for example, temperature sensitive biological materials, such as live cells, cell extracts, monoclonal antibodies, and like materials. The freezer system and temperature and rate-controlled cooling methods of the disclosure are also generally applicable to non-biological materials and processing, such as semi-conductor fabrication, polymorph or crystallization, magnetic materials, conductive materials, and like applications.
The present disclosure, in embodiments, provides a freezer system including a temperature monitored and temperature controlled freezer characterized by being capable of producing and maintaining uniform temperatures within individual samples and uniform temperatures among samples. The freezer system is also characterized by being capable of producing constant rate-controlled cooling within individual samples and among samples. The freezer system is also characterized by being capable of minimizing the temperature differential among samples and the temperature differential between samples and the chamber. The minimized temperature differentials are believed to enable more uniform sample temperatures and more uniform sample cooling-rates. Thus, the freezer system can provide superior cryo-preserved samples having little or no lot-to-lot variability in sample temperature, relative cooling-rate, and sample quality or efficacy.
The present disclosure, in embodiments, also provides a rack system comprising one or more trays adapted to receive and hold items, such as sample vials or ampoules, within the freezer to further enable the above-mentioned temperature and cooling characteristics of the freezer system and cooling methods of the disclosure. The freezer system incorporating the rack system is characterized by being able to accommodate a plurality of same or different sized items. The freezer system incorporating the rack system is further characterized by having an increased total sample volume or sample load capacity for simultaneously and rate-controllably cooling items compared to commercially available freezers.
Accordingly, in embodiments, the present disclosure provides:
a freezer system including a freezer, temperature measurement and temperature control system, and a rack system, which freezer system is adapted to provide rate-controlled cooling and optionally preservation of temperature-sensitive items;
a rack system including a tray adapted to receive and hold a plurality of items in the freezer system of the disclosure;
a tray adapted to receive and hold a plurality of items, adapted to permit stackable assembly of two or more trays, and adapted to disturb laminar refrigerant flow patterns within the freezer system of the disclosure;
a method for rate-controlled cooling of a plurality of items in the freezer system of the disclosure;
a method for rate-controlled freezing of a plurality of items in the freezer system of the disclosure; and
a method for preserving an item in the freezer of the disclosure employing rate-controlled cooling methods of the disclosure.
These and other embodiments are illustrated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic plan of a freezer system in embodiments of the present disclosure.
FIGS. 2A and 2B illustrates a cross-sectional view of a freezer (2A), and a cross-sectional view of a tube (2B), in embodiments of the present disclosure.
FIGS. 3A and 3B illustrates a cross-sectional (3A) view and a perspective (3B) view of a rack system for use in a freezer system in embodiments of the present disclosure.
FIG. 4 illustrates a plan view of an unfilled tray for use in the rack system and a freezer system in embodiments of the present disclosure.
FIG. 5 illustrates a plan view of a partially filled sample tray having thermo-sensor wiring for use in the rack system and freezer system in embodiments of the present disclosure.
FIG. 6 illustrates a plan view of an alternative configuration of a partially filled sample tray for use in the rack system and freezer system in embodiments of the present disclosure.
FIG. 7 illustrates a plan view of another alternative configuration of a partially filled sample tray for use in the rack system and freezer system in embodiments of the present disclosure.
FIG. 8 illustrates a chart of exemplary time versus temperature rate-controlled cooling achieved with an appropriately partially filled tray in a freezer system in embodiments of the present disclosure.
FIGS. 9A and 9B illustrates a cross-sectional view of a stackable tray of a rack system in embodiments of the present disclosure.
FIG. 10 illustrates a perspective view of a stackable tray of a rack system in embodiments of the present disclosure.
FIG. 11 illustrates a perspective view of another stackable tray of a rack system in embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present disclosure provides the abovementioned freezer system, rack system, stackable tray article, and methods for their use.
Basic components and operation thereof of a commercially available rate-controlled freezer are disclosed, for example, in the above-mentioned U.S. Pat. No. 6,044,648. Rate-controlled freezers having a chamber, circulating fan and plenums, evaporating coil or tube, refrigerant source, regulating value, and solenoid, are commercially available and several of these methods were used and evaluated, in embodiments, in constructing the freezer systems, components, and methods, of the present disclosure.
The present disclosure provides a cooling device generally including a housing having a chamber and an air circulation path at least partially within the chamber. A fan is operatively connected to the chamber for circulating air within the air circulation path. In accordance with a general aspect of the disclosure, a perforated coil or tube is disposed within the air circulation path of the chamber and receives a liquid refrigerant, such as liquid nitrogen, or the like refrigerants, under pressure. Preferably, the tube is formed as a coil having at least one revolution and, more preferably, a plurality of revolutions. The perforations can be disposed at least along an inwardly facing surface of the coil and air is circulated through the coil by the fan. In embodiments, the fan can be an axial blade fan and the coil is disposed generally about the fan. It will also be appreciated that the coils in these embodiments may be spaced apart. In embodiments, the coil can be disposed generally about the fan. However, the fan in this case is a centrifugal fan. In this latter embodiment, the perforated tube can preferably be formed as multiple, spaced apart coils and the centrifugal fan directs air between the spaced apart coils during the cooling process.
The present disclosure improves the uniformity of sample item or product cooling by providing sample trays, which disturb or disrupt laminar refrigerant-air flow patterns within the chamber. This permits the transfers a large percentage of the sample item heat to the refrigerant through convection and by mixing the circulating process air with cold refrigerant vapor. In the present disclosure, the liquid refrigerant, such as liquid nitrogen, is injected into a coiled, perforated tube of one or more turns which tube is used as a shroud over the process air fan. The liquid refrigerant is injected at a higher pressure, preferably 22-40 psig, than the process air in the chamber, which is for example typically at 0 psig or ambient atmospheric pressure. This results in the liquid refrigerant entering the coil with enough velocity to centrifugally force the liquid refrigerant against the outermost portion of the inner wall surface of the coiled tube. The heat from the process air within the chamber is transferred to the coil by means of the forced convection. As the liquid refrigerant expands and absorbs heat, it is converted to cold refrigerant gas vapor. This gas separates from the liquid by virtue of greatly reduced density, and is expelled from the coil through a series of holes or perforations, for example, on the inside diameter of the coiled tube. This cold refrigerant gas mixes with the process air by the process air fan, further cooling the process air. The process air, cooled by the above mechanisms, absorbs heat from the items or samples in the tray or rack system to uniformly cool the sample items within the chamber.
The present disclosure can further improve the uniformity of sample item cooling by process control, for example, providing refrigerant flow-rate controls such as active controls on the refrigerant, such as admission and evaporation rate; and passive controls on the refrigerant such as circulation path and patterns, with for example, a fan speed controller, a baffle, and optional vent adapted to controllably admit or expel warm gas, e.g., ambient air, ambient nitrogen, or mixtures thereof, from an external source into the housing, and preferably to the chamber. Additionally or alternatively, the above-mentioned vent can be used to controllably expel gas or gas mixtures from the chamber or housing. In embodiments, the vent can be, for example, a manually or preferably a computer controlled valve permitting either or both venting the chamber to atmosphere and admitting a second gas, such as ambient temperature air, nitrogen, or both. The vent can be used to regulate, as needed, ingress of ambient temperature gas or egress of gas or gas mixtures within the chamber to balance or counter-act, for example, localized excessive cooling excursions.
Highly uniform cooling-rates and temperature control is also enabled by employing multiple temperature monitors or thermo-sensors, such as digital or analog, adapted to sense and measure the temperature in the chamber, and optionally adapted to control or regulate the temperature in the chamber, for example, by altering, such as by accelerating or decelerating, the refrigerant flow-rate into the chamber; by altering, such as by accelerating or decelerating the fan speed or revolutions per minute (rate), which can change the circulation rate of the refrigerant gas mixture within the chamber; by altering, such as by accelerating or decelerating, the ingress flow-rate of ambient gas into the chamber; or by altering, such as by accelerating or decelerating, the egress flow-rate of chamber gas out of the chamber to ambient air.
The following definitions are used, unless otherwise described.
“Baffle” refers to a structural element within the chamber, which baffle can alter the refrigerant air-flow patterns in the chamber compared to when the baffle is absent. A baffle can be attached, for example, to any part of a tray, to any part of the rack system, to any part of the interior of the chamber, or combinations thereof. Although not desired to be limited by theory, a baffle is believed to alter the refrigerant air-flow patterns in the chamber compared to when the baffle is absent, for example, resulting in greater non-laminar flow and lesser laminar flow, to thereby provide for superior uniform cooling-rate and temperature control of sample items situated in the chamber. Although not wanting to be limited by theory, it is believed that the baffle can favorably disrupt the typical laminar refrigerant flow patterns that occurs in the abovementioned commercially available freezer models to produce non-laminar refrigerant flow patterns. These non-laminar refrigerant flow patterns, in embodiments, are believed to provide the aforementioned improved temperature and rate-controlled cooling properties of the disclosure.
“About” modifying, for example, temperatures, times, cooling rates, flow rates, pressures, dimensions, and like values, and ranges thereof, employed in the disclosure, refers to variation in the numerical quantity that can occur, for example, through typical measuring and processing procedures used in making and using the invention; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make and use the compositions, apparatus, or methods; and the like variations. Whether or not modified by the term “about” the claims include equivalents to the quantities.
As used herein, “consisting essentially of” refers to the articles, devices, or methods, which can include the components listed in the claims, plus other components that do not materially affect the basic and novel structure or operation of the articles, devices, or methods.
The indefinite article “a” or “an” and its corresponding definite article “the” as used herein is understood to mean at least one, or one or more, unless specified otherwise.
In embodiments of the present disclosure there is provided an apparatus and methods for controlled-cooling of items. The controlled-cooling of items can be accomplished in embodiments of the disclosure by, for example: structures which disturb or alter otherwise laminar refrigerant flow, such as a baffle or an aperture; structures which promote or provide alternative pathways for greater or more efficient circulation of non-laminar refrigerant flow, such as a baffle or an aperture; close monitoring and control of temperature to minimize temperature differentials between the apparatus and the cooled items and minimize temperature differentials among the cooled items, and combinations thereof.
In embodiments of the present disclosure, the freezer system can be used in cooling, freezing, or preserving sensitive biological materials.
In a preferred embodiment, the freezer system can be used in combination with other known cooling equipment and preservation methods to achieve excellent and reproducible cooled samples.
It will be readily apparent to one of ordinary skill in the art that larger volume samples and their containers have a smaller surface-to-volume ratio and are more difficult to uniformly cool, that is, where the immediate surrounding or out-side temperature is comparable to the inside temperature, compared to a relatively smaller volume sample containers which have a higher surface-to-volume ratio.
Thus, in preferred embodiments sample items having relatively larger volumes, such as 5 to about 100 mL each, can be rate-controllably cooled at considerably greater total volumes and comparable rates to sample items having smaller volumes, such as 0.1 to about 1 mL, and in a freezer system other than those of the present disclosure.
In embodiments the present disclosure provides a freezer system comprising:
a freezer adapted to cool items;
a thermo-sensor to monitor the temperature within the freezer;
at least one tray having apertures to hold the items within the chamber of the freezer and to provide, that is to facilitate, uniform cooling of the items;
a programmable controller to receive and process signals from the thermo-sensor and to control the temperature of the freezer and the cooling-rate of the items; and
a baffle within the freezer.
In embodiments, a baffle can be part of a tray. In embodiments, a baffle can be preferably situated so that the baffle disturbs laminar refrigerant flow and causes turbulent refrigerant flow and thereby facilitates uniform cooling of the items.
Programmable controllers or programmable logic controllers, temperature controllers, temperature sensors, servo-controllers, servo-motors, and like gear, are commercially available, see for example, the “Allen-Bradley Controllers” website <http://www.ab.com/plclogic>, RadioShack™<www.radioshack.com>, and like vendors, and can be readily adapted or modified for use in embodiments of the present disclosure.
The freezer system can further comprise at least one tray adapted to hold the items within the chamber of the freezer. The at least one tray can comprise from two to five trays. In embodiments, the rack system can comprises from 1 to about 10 trays, such as 2 to about 10 trays, preferably from 2 to 7 trays, more preferably from 3 to 5 trays. In embodiments, the tray can have one or more baffles associated therewith, for example, attached to or affixed to the tray or integral to the tray, such as molded into or sculpted from the tray structure. In embodiments, the trays, rack system, and freezer system can accommodate many items. For example, each tray can accommodate from 10 to about 200 items.
The thermo-sensor(s) can monitor the temperature of one or more locations on a tray, and can monitor the temperature of one or more of the items situated in a tray. For example, the thermo-sensor can be a thermocouple and the thermocouple can be in contact with the exterior of a sample ampoule. Alternatively or additionally, the thermo-sensor or thermocouple can be inside the sample ampoule and optionally in direct contact with the sample material. In embodiments, at least one thermo-sensor is situated within the chamber of the freezer and away from the freezer's refrigerant source, for example, the fan and coil refrigerant evaporator, to avoid false or artificially low temperature(s) readings and to better reflect the ambient temperature near the sample items being cooled.
In embodiments the programmable controller can signal a cooling-rate of from about −0.1 to about −10° C. per minute, for example, in accordance with a cooling rate program. A preferred cooling-rate program signals a cooling-rate of about −0.1 to about −1° C. per minute. Another preferred cooling-rate program signals a cooling-rate of from about −1° C. plus or minus 0.2° C. per minute over a temperature range of from about 40 20 C. to about −100° C. To control the temperature and the cooling-rate the programmable controller can, for example, regulate the evaporator fan speed, regulate the flow rate of an external source of liquid refrigerant into the chamber, regulate the flow rate of air vented from circulating within the chamber to outside the housing, regulate the orientation of at least one baffle, and combinations thereof.
In embodiments the programmable controller can also be adapted for controlling, for example, the size, orientation, effectiveness, or like properties of a baffle or an aperture in disrupting or re-directing the coolant-air mixtures within the freezer system. Thus, for example, a plurality of unoccupied apertures of a tray (e.g., sheet or sidewall) can be fitted, or manufactured, with one or more aperture valve assemblies (known in the photographic arts and elsewhere), one or more guillotine value assemblies (e.g., an adjustable, such as slidable, sheet attached to a tray sheet which can be slidably adjusted to regulate the size of the aperture(s)), or like equivalents for modifying the dimensions of the apertures, and having a servo-motor or an equivalent mover to reversibly open and close the valve. The valve assemblies can be optionally connected to the programmable controller and can be selectively or collectively used to further regulate circulation of the coolant-air mixtures to achieve optimal cooling temperatures and cooling rates, such as, an initial rapid cooling which transitions to a gradual or slow cooling. In another example, in embodiments where a baffle is, for example, a moveable panel member, such as a reciprocating louver, a moveable or modifiable panel member, or like configurations, the movement, orientation, location, and like aspects of the baffle can be accomplished or modified with, for example, a mechanical or pneumatic drive or like equivalent mover, and which mover can be optionally connected to and controlled by the programmable controller. It will also be appreciated and as illustrated herein, that a baffle can have one or more apertures there through. In embodiments, a baffle, an aperture in the baffle, an aperture in a tray structure or like structures of the freezer system, or combinations thereof, and further in combination with a servo-motor or similar equivalent mover can be connected to the programmable controller and can be used to selectively or collectively further regulate circulation of the coolant-air mixtures to achieve optimal cooling temperatures and cooling rate profiles.
In embodiments the present disclosure provides a freezer system comprising:
a freezer comprising a housing having a cooling chamber therein;
a rack system comprising one or more trays in the chamber, the trays being adapted to receive and hold items for cooling;
at least one baffle associated with the rack system or chamber; and
a temperature controller which can measure temperatures within the freezer, control the temperature, and provide a constant cooling-rate for the items in response to the measured temperatures.
The freezer system provides uniform temperature and rate-controlled cooling of the items in the freezer. In embodiments the freezer can further comprise a liquid refrigerant source, a valve and solenoid adapted to regulate the liquid refrigerant admitted to the housing, a coil adapted to vaporize the liquid refrigerant, and a fan adapted to circulate the vaporized refrigerant through the chamber. The temperature controller or control system can comprise a plurality of thermo-sensors adapted to measure, for example, the temperature of the chamber, the rack system, and items placed therein, such as multiple for temperature monitoring and control which thermocouples are positioned and distributed within the chamber, on or within the rack, on or within a tray, and on or within the sample items. The temperature controller can be adapted to control the solenoid, which regulates the liquid refrigerant valve, adapted to control the fan speed, or adapted to control the circulation rate of the air-refrigerant mixture within the chamber.
In embodiments the rack system can comprise one or more trays where each tray can be adapted to receive and hold a plurality of items and as illustrated herein. In embodiments the rack system can comprise one or more trays where each tray can be adapted to stackably engage another tray, for example, stack one or more trays upon one or more other trays to form a self-supporting rack system and without the need for an additional frame support member and as illustrated herein. In embodiments the rack system can comprise a frame adapted to slidably receive one or more trays analogous to drawers in a dresser.
In embodiments the rack system can be adapted to slidably engage the chamber, for example, in a drawer-like fashion. In embodiments the rack system can be adapted to provide an electrical connection to the temperature controller when the rack system or one or more trays are slidably engaged in the chamber. Thus, the chamber and rack can be equipped with wires and connectors so that they can be easily connected to the temperature monitoring and control electronics outside the freezer housing. In embodiments the rack system can be adapted to slidably engage one or more trays inserted into the rack system. In embodiments the rack system can be adapted to provide an electrical connection between the temperature controller and one or more trays when the tray(s) is (are) inserted into the rack system or trays, for example, when a tray is electrically plugged into the rack. The rack system of the disclosure having integral wiring and connectors provides for greater safety for operators and for sample items, and greater operator and operational efficiencies, for example, in loading and unloading trays, and unloading samples from trays.
The freezer system can provide a relatively constant cooling-rate of about −1 degree centigrade per minute over from about 35° C. to about −90° C., for example in to about −90° C. The freezer system provides a low temperature variation within an item of less than, for example, from about 0.01 to about 0.5° C., measured for example, side-to side, side-to-center, top-to-bottom, or bottom-to-middle. The freezer system can provide a low temperature variation between or among two or more samples of less than, for example, from about 0.1 to about 5.0° C. The freezer system can provide a low temperature variation between any two locations in the chamber, rack system, tray, or sample item, of less than, for example, from about 0.1 to about 5.0° C.
Although not desired to be limited by theory, in embodiments the rack system perturbs linear or laminar air-flow patterns in the chamber. The perturbation of laminar air-flow in the chamber is believed to produce a more random, albeit even distribution, pattern of cool air flow to provide highly uniform temperatures and sample item cooling-rates. In embodiments the rack system can further comprise one or more trays where the trays can have one or more baffle members attached to the trays. In preferred embodiments each tray has a baffle at both ends and perpendicular relative to the laminar air-flow pattern found in the freezer chamber in the absence of the trays with baffles. In embodiments, an interior wall of the chamber can include a baffle if desired. In embodiments a baffle can be, for example, a fixed panel member, an adjustable panel member, a moveable panel member, a modifiable panel member such a pluggable baffle described below, or plural movable panel members, such as a mechanically engaged or pneumatically driven reciprocating louver, for example, situated between the fan and the chamber, and like arrangements. In embodiments, when the baffle is attached to the end(s) of a tray, the baffle can be, for example, half the height of the tray, the full height of the tray, intermediate heights between half and full height, heights less than half height, heights greater than full height, and like heights. The tray height can be the separation between a top most sheet and a bottom most sheet when no legs, pegs, or other tray structure extends beyond the sheets. The tray height can be the separation between a top most tray structure and a bottom most tray structure, such as a leg, peg, connector, baffle, or other tray structure which extends beyond, above or below, the tray sheets.
The present disclosure in embodiments provides a tray comprising:
a base sheet adapted to support items, the base sheet having an optional array of apertures there through, the base sheet apertures when present being adapted to be vacant to permit cross-flow refrigerant circulation in a freezer;
a top sheet having an array of major and minor apertures there through, the top sheet array of major and minor apertures being adapted to be non-collinear with the optional array of apertures of the bottom sheet, the each major aperture being adapted to either hold a item or to be vacant and to permit cross-flow circulation of circulating refrigerant in a freezer, the array of minor apertures being adapted to be vacant and to permit cross-flow refrigerant circulation in a freezer; and
a connector adapted to fixably join together and space apart the adjacent sheets of a tray from each other, and optionally adapted to permit two or more trays to be securely stacked together.
In embodiments of the present disclosure, the tray can further comprise a baffle connected to the tray. In embodiments, a baffle can be connected to at least one of the tray sheets. In embodiments, two baffles can be connected to opposite ends of sheets of the tray, and the tray ends or sheet ends can be oriented in the main flow path of circulating refrigerant in a freezer.
In embodiments of the present disclosure, the connector assembly joining tray sheets together to form a tray can, for example, comprise a baffle. In this and other embodiments of the present disclosure, a baffle can optionally have apertures there through to, for example, obtain substantial deflection, diversion, or regulation of the main or ancillary air-flow pattern(s), yet selectively creating one or more air channels into and through a tray assembly. In embodiments the spacer can be, for example, a wooden dowel and the baffle can be a cardboard sheet. In embodiments the spacer can be, for example, a joinable hollow aluminum or other metal or metal alloy and the baffle can be an aluminum or other metal or metal alloy sheet. In embodiments each tray can have at least one baffle attached thereto. In embodiments each tray can have at least two baffles attached thereto. In embodiments multiple trays can have combinations of a tray having one baffle attached thereto and another tray having two or more baffles attached thereto, depending for example, on the type of cooling desired, sample identity and volume, the type and placement of baffles on the trays, such as tray-end baffles, sheet baffles, and like considerations.
In embodiments, the connector can further comprise a leg member, for example, adapted to elevate a tray base plate or sheet to afford an air-way beneath and traversing the base plate and optionally adapted to permit tray stacking. A connector rod or a spacer member, when properly proportioned can provide the leg member. Additionally or alternatively, a vertically oriented peg or pin can be attached to, or integral with an end baffle member, which peg or pin can be adapted to interlock or mate with, for example, an aperture in an adjacent (i.e., above or below) stacked tray and as illustrated herein.
In embodiments, the connector or the connector assembly can comprise, for example, two baffles connected to opposite ends of the tray, the ends being oriented in and perpendicular to the main flow path of circulating refrigerant in the freezer. In embodiments, the connector assembly can further comprise a peg member adapted to elevate the tray base plate or sheet of the tray to afford an air-way beneath and traversing the base plate and optionally adapted to permit tray stacking and as illustrated herein.
In embodiments, the connector can comprise a connector rod, an optional spacer, an optional nesting washer, and an optional fastener. In embodiments, the connector rod can comprise a first leg member adapted to fasten, such as into a screw hole or welded, into the bottom of the base sheet, and a second spacer member adapted to fasten, such as into a screw hole or welded, between the base sheet and an upper sheet, such as a top sheet or optional middle sheet. In embodiments, an optional nesting washer can be adapted to stack and electrically interconnect two or more trays, for example, to receive, hold, and connect, the leg of another tray to the top of lower tray. Similarly, the connector can be further adapted to permit electrical interconnection of the tray to another tray and to permit electrical connection of the tray assembly or rack system to a temperature measurement system. The electrical connections can be used to connect the thermo-sensors to the temperature controller. Additionally or alternatively, the electrical connections can be used to connect, for example, resistive heater elements to the temperature controller to be used in, for example, controlling temperature excursions or in rate-controlled warming or temperature moderation processes.
It is also readily appreciated that the abovementioned electrical connections between the controller and the thermo-sensors, solenoid, and like components can be omitted or reduced by, for example, substituting with wireless equivalents and having appropriate transmitter and receiver means, such as the commercially available BLUETOOTH™ technology set, see for example <http://www.bluetooth.com>.
In embodiments, the tray can further comprise an optional middle sheet interposed between the top sheet and bottom sheet, the middle sheet having an array of major and minor apertures there through which are identical and collinear to those apertures through the top sheet.
The present disclosure in embodiments provides a rack system comprising:
an above mentioned tray adapted to receive and hold items, such as sample vials or ampoule. In embodiments, the rack system can comprise, for example, 1 to 5 trays or more. The rack system can include a connector assembly, which can comprise a baffle, the baffle optionally having apertures there through. The rack system comprising a tray can hold a plurality of items, for example, from 60 to 150 items. An item can comprise, for example, a glass ampoule having a sample volume of from about 5 to about 150 mL. A tray loaded with items in a pattern can have, for example, a total sample volume capacity of from about 1,000 to about 15,000 mL. The glass ampoule items can contain, for example, a biological material such as live cells, including plant, animal, or like cells, tissue specimens, cell extracts, antibodies, viruses, and like materials. If desired, the rack system can further comprise an optional shaker adapted to controllably shake the rack or tray and agitate the items in the tray to, for example, further facilitate uniform cooling of the items, and optionally to mix or keep sample specimens suspended in a liquid matrix.
The present disclosure in embodiments provides a method of cooling with an above mentioned freezer system,
the freezer system comprising:
a freezer adapted to cool items;
a plurality of thermo-sensors adapted to monitor the temperature at points within the freezer;
a programmable controller adapted to receive and process signals from the thermo-sensors and further adapted to send signals to control the temperature and the cooling-rate of the items in the freezer; and
a tray comprising:

- a base sheet adapted to support items, the base sheet having an optional array of apertures there through, the base sheet apertures when present being adapted to be vacant to permit cross-flow refrigerant circulation in a freezer;
- a top sheet having an array of major and minor apertures there through, the top sheet array of major and minor apertures being adapted to be non-collinear with the optional array of apertures of the bottom sheet, the each major aperture being adapted to either hold a item or to be vacant and to permit cross-flow circulation of circulating refrigerant in a freezer, the array of minor apertures being adapted to be vacant and to permit cross-flow refrigerant circulation in a freezer;
- a connector adapted to fixably join together and space apart the adjacent sheets of a tray from each other, and optionally adapted to permit two or more trays to be stacked together; and
- optionally a baffle connected to the end of the tray; the method comprising:

arranging a plurality of items in a pattern in the tray;
placing the tray in the freezer's chamber and sealing the chamber;
circulating the air and a gaseous refrigerant through the chamber and in contact with the items;
monitoring the temperature at points in the chamber with the thermo-sensors; and
controlling the temperature and the cooling-rate of the items in the freezer with the programmable controller.
In embodiments, the pattern of items in a tray can comprise having an item occupy an aperture, for example, either major or minor apertures, and having any adjacent apertures vacant, that is, where a sample item residing in a aperture in the tray has no nearest neighbors.
In embodiments, the pattern of items in a tray can comprise having major apertures on the periphery of the air-flow channel of the tray occupied with items and having major apertures proximal to the air-flow channel of the tray vacant or free of items.
In embodiments, the cooling-rate can be, for example, about −1° C. per minute, for example, over a temperature range of about +40 to −100° C. In embodiments, the method can provide uniform temperatures within individual samples and uniform temperatures among items. In embodiments, the method can provide a narrow temperature differential among items. In embodiments, the method can provide rate-controlled cooling within individual samples and among items. In embodiments, the method can provide a narrow temperature differential between the chamber and the items in the chamber. In embodiments, the controller can control the temperature and cooling-rate of items by, for example, regulating the amount refrigerant admitted to the chamber and the circulation rate of the air and refrigerant in the chamber, such as by operating a circulating fan at a faster or slower speed in response to the measured temperatures, and in accordance with the predetermined cooling-rate program and desired temperature profile.
It will be readily appreciated by one of ordinary skill in the art that the freezer system and components thereof, and cooling methods thereof of the disclosure can be used to cool sample items in a rate-controlled manner and thereafter to store the resulting cooled preserved sample items. In embodiments, the freezer system and components thereof, and cooling methods of the disclosure can be used to cool samples in a rate-controlled fashion and the resulting cooled samples can there after be transferred, for example, in a rack system, in a tray of the disclosure, or individual samples, to a dedicated low temperature high capacity storage freezer, such as a freezer chest or “walk-in” freezer unit having larger storage capacity and at least constant temperature controls. It is also readily apparent from comprehending the present disclosure that the cooling system, components, and methods of the disclosure can be readily adapted for use in larger sized freezer systems such as a “walk-in” freezer unit. It will also be readily evident to one of ordinary skill in the art that the freezer system and its components, and the cooling methods of the disclosure can be used, if desired, to rate-controllably warm or thaw sample items, for example by accomplishing a rate-controlled cooling routine or program of the disclosure in reverse, that is, from lower temperatures to higher temperatures.
Referring to the Figures, FIG. 1 illustrates a schematic plan of a freezer system (100) in embodiments of the present disclosure, including a freezer housing (110) which encloses a freezer chamber (120). The chamber in embodiments, is sealable and can accommodate a rack system which can comprise, for example, a rack frame and one or more trays (140), such as from 1 to about 5 trays, supported by the rack frame, and adapted to receive and hold items (145) such as sample ampoules, or like items. In embodiments a frame is not necessary and appropriately configured trays when combined can be self-supporting and as illustrated herein. Attached to the freezer is a source of compressed liquid refrigerant (150) such as liquid nitrogen, which admission to the housing is regulated by, for example, a valve (160), and which valve can be optionally remotely controlled by, for example, a solenoid (170) or like devices. For example, the solenoid (170) can be subject to programmable control and regulation by communication with a programmable controller (180). The controller can interactively provide temperature monitoring and rate-controlled cooling and as illustrated herein. The controller also preferably is connected to and processes signals obtained from thermal sensors, for example, thermocouples or like devices, such as one or more “in-sample” sensors (181), “in-chamber ” sensors (182), or “in-tray” sensors (183). The freezer system can also optionally include a computer (190) for convenient display, data-logging, computation, networked communication, and like enhanced features of basic programmable control functions.
FIG. 2A illustrates a cross-sectional view of a freezer (100), which comprises in embodiments a housing (212) having an interior chamber (214) formed by interior walls (216, 218). Interior walls (216, 218) preferably also form respective upper, lower and side air plenums (220, 222, 224) which, together with chamber (214), form an air circulation path (e.g., arrows shown). A fan (226) is disposed within this air circulation path and is connected to a thermally isolated motor, as shown, to move air within chamber (214), plenums (220) and (222), and finally back into plenum (224) in which fan (226) is disposed to complete the refrigerant-air circulation. In accordance with the disclosure, a tube (228), preferably in the form of a coil having perforations (230), is disposed within the air circulation path. Specifically, coiled tube (228) is disposed within plenum (224) and circularly about fan (226). Coil (228) is further disposed about a opening (231) contained in chamber forming walls (216, 218) of the housing enclosure (212). As shown, fan (226) can be disposed in alignment with this hole (231). A source of liquid refrigerant, such as liquid nitrogen (232) is connected to an open end (228 a) of coil (228), preferably via a valve (234) operated by a solenoid (236). Another end (228 b) of coil (228) is closed. Perforations (230) preferably can be, for example, about ⅛″ in diameter and spaced about 4 inches apart; however, this may vary according to the application.
FIG. 2B is an enlarged cross-sectional view of a coil (228), in embodiments of the present disclosure, showing liquid refrigerant (238) and is injected at a pressure of about 22-40 psig and preferably flows along an outside portion of tube (228) through centrifugal force. This liquid refrigerant, such as liquid nitrogen, will vaporize as it travels through the coil and the vaporized gas will exit the tube through apertures (230) as shown in FIG. 2A. Thus, this cold gas will exit into the air flow created by fan (226) and will uniformly flow into chamber (214) to uniformly cool the product contained therein. Ideally, all of the refrigerant in liquid phase will vaporize through coil apertures (230) as it travels through coil (228) before reaching a closed end (e.g., 228 b).
FIG. 3A illustrates a cross-sectional view of a drawer rack system for use within the freezer chamber (305, not shown), in embodiments of the present disclosure, including a frame rack (300), having optional leg supports (302), which frame is adapted to slidably receive, support, and hold one or more sample trays (310)(three shown). The trays, in embodiments, can be comprised of a top plate (310 a), an optional middle plate (310 b), and a lower plate (310 c), and adapted to receive and hold a plurality of items (320), such as sample ampoules, for rate-controlled cooling. In embodiments, the trays are each supported in the frame by cross-shelf members (325). The tray plates can be held together, stabilized, and separated by, for example, connector-spacer members (315) or like joining fasteners. In embodiments a tray can have one or more baffle plates (317).
FIG. 3B illustrates, in embodiments of the present disclosure, a perspective view of the drawer rack system of FIG. 3A, but without trays present, including a frame rack (300), having optional leg supports, showing a closed or mesh top sheet (330), a closed or mesh bottom sheet (335), fan-side or upwind-side mesh wall (340), open walls (345)(back, front, and down-wind side), optional thermo-sensor plug-in and connections (350) adapted to connect the frame wiring to the freezer and an external controller, and optional thermo-sensor wiring and connections adapted to electrically connect one or more trays to the frame wiring (360).
FIG. 4 illustrates, in embodiments of the present disclosure, a plan view, for example, as viewed from above, of an unfilled tray (400) showing the relative distribution of apertures or openings, for example, in the top and optional middle tray sheets or plates. In embodiments, the sheet openings can be the same diameter. In embodiments, the sheet openings preferably can be of different diameters, for example, a larger array or major set and a smaller array or minor set, to enable a human operator or a robotic sample loader to readily discern different diameter apertures and sample item locations, and to accord preferred sample item distributions or sample load configurations. Thus, for example, there can be major openings (410) having larger diameter openings and there can be staggered minor openings (420). The above-mentioned tray plate spacer-connector member (430) having, for example, a top-side location can be disassembled for cleaning or storage, and readily checked and securely maintained by an operator. In embodiments, there can be, for example, from about 5 to about 20 major openings (410) (ten shown) to an across-side (left-to-right) and from about 5 to about 20 major openings (410) (nine shown) up-side (bottom-to-top), and from about 5 to about 20 minor openings (420) (nine shown) to an across-side (left-to-right) and staggered between the major openings and from about 5 to about 20 minor openings (420) (eight shown) up-side (bottom-to-top). This opening distribution provides a hole or aperture matrix of 90 major openings (410) and 72 minor openings (420) for a total of 162 openings for various combinations of samples and vertical or cross-flow circulation patterns. In comparative examples, when the tray's major openings (410) were all occupied with sample items and there were no minor openings (420), there appeared an undesirable “warm-zone” (450) within which samples items were not frozen and while sample items outside the zone were frozen.
It will be readily apparent to one of ordinary skill in the art that the plate openings, if desired or not required, can be identical or similar in diameter. It will also be readily apparent to one of ordinary skill in the art that there should be no plate openings in the bottom plate so as to prevent samples from inadvertently falling-through and resulting in breakage during loading. Alternatively, if plate openings are present in the bottom plate or sheet, they are preferably smaller in diameter relative to the sample item diameter to avoid the breakage problem yet still allow for cross-flow circulation of the refrigerant air, preferably the base sheet openings are not collinear with upper sheet openings, or both smaller and not collinear.
In embodiments, the minor openings (420) can be purposely left vacant, that is without a sample item present in the minor openings, which vacant openings can serve as vertical channels which provide enhanced circulation of the refrigerant air and improved heat-exchange between the sample items which may be present in the major openings (410). In embodiments, for example where smaller sample items are used and are better accommodated (i.e., fitted) in the minor openings (420), the roles can be reversed and the major openings (410) are vacant and can serve as vertical channels which provide enhanced circulation of the refrigerant air and improved heat-exchange between the sample items in the minor openings (420).
FIG. 5 illustrates in embodiments of the disclosure, a plan view, as viewed from above, of a partially filled sample tray (500) of FIG. 4, where the sample item fill- or distribution-pattern provides that each of the major openings (510) is filled with a sample item (530) such as a sealed glass ampoule, a screw-cap plastic ampoule, and like containers, so that each major opening (510) also has a nearest-neighbor major opening (510) which is also filled with a sample item and where all minor openings (520) are vacant and provide vertical channels for refrigerant-air circulation. In embodiments, for example, where the sample loading (total volume) is large or maximized, the sample loading configuration of FIG. 5 may be less preferred compared to other configurations for optimal rate-controlled cooling as mentioned above, i.e., a warm-zone, and as illustrated herein. In embodiments, a tray plate or tray sheet as shown in FIG. 5, such as top, middle (if present), or bottom plate of FIG. 3, can optionally be wired (555) with thermo-sensors (560) which enable modular connection of the tray wiring (550) to the abovementioned rack frame wiring, or alternatively, at least permitting connection to or communication with the external temperature controller as suggested and discussed in FIG. 9B below. Sensors (560) can be attached to or embedded in the tray surface at various locations or levels, attached to ampoules, inserted within the ampoule or simulated ampoules, or combinations thereof. In embodiments, where trays without baffles (as discussed below) were used and filled with ampoules as shown in FIG. 5, uneven, incomplete, warm-zone, or similar gradient cooling was observed. The sample item configuration can have significantly improved uniform sample cooling rates when the tray is out-fitted with one or more baffle members as discussed below.
FIG. 6 illustrates in embodiments of the disclosure, a plan view, as viewed from above, of a partially filled sample tray (600) of FIG. 4, where the sample item (605) and the fill- or distribution-pattern of a plurality of the sample items provides items that alternate, or “every-other” of the major openings is filled (610) with a sample item (605), such as a sealed glass ampoule, so that no filled major opening (610) has a filled nearest-neighbor major opening nor any filled minor opening nearest-neighbors. Thus, each filled major opening (610) is occupied with a sample item and its adjacent or nearest neighbor major (615) and minor (620) openings, are vacant to provide vertical channels for enhanced refrigerant-air circulation. In embodiments, a tray plate or tray sheet as a shown in FIG. 6, such as top, middle (if present), or bottom plate of FIG. 3, can optionally be wired (650) with thermo-sensors (not shown, reference FIG. 5) which enable modular connection of the tray wiring to the abovementioned rack frame wiring. In embodiments, the tray can include an optional baffle (660, 670) attached to one or both ends of the tray assembly, by for example, press fit (shown), fasteners, such as a screw, a weld, a low-temperature insensitive adhesive, or like fastening means or methods. In embodiments when samples were cooled in trays having one or both baffles (660, 670) present and oriented perpendicular or traversing the main coolant current or coolant source flow pattern (e.g., 680), superior uniform convective cooling of samples was observed. The superior cooling uniformity observed with the baffle(s) present is believed to be due to the disruption or disturbance in the coolant flow pattern into smaller more diffuse flow patterns (e.g., 690) upon contact of the main current flow (680) with the lead-edge baffle (660), which diffusion circumvents the baffle, above, below, and around the ends of the baffle.
FIG. 7 illustrates in embodiments of the disclosure, a plan view, as viewed from above, of a partially filled sample tray (700) of FIG. 4, where the sample item (705) and the fill-pattern or distribution-pattern of a plurality of sample items provides, for example substantially as shown, two peripheral and parallel rows of the major openings are, for example, entirely filled (710) with a sample items, and the fill-density of unfilled major openings (715) and unfilled minor openings (720) is reduced toward the center of the tray. The lateral center (left-to-right) of the tray corresponds approximately to, in embodiments, a major laminar refrigerant flow pathway. The unfilled or vacant openings provide vertical channels for enhanced refrigerant-air circulation. In embodiments, a tray plate as a shown in FIG. 7, such as top, middle (if present), or bottom plate, of FIG. 3, can optionally be wired with thermo-sensors (750) which enable modular connection of the tray wiring and the sensors, such as thermocouples, to the above mentioned optional rack frame wiring and external to the freezer, such as to the controller. In embodiments, the tray can include an optional baffle (760) attached to one or both ends of the tray assembly, by for example, press fit (shown), fasteners, such as a screw, a weld, an adhesive, or like fastening means or schemes. The baffle(s) (760) can include optional baffle aperture(s) (e.g., 765). In embodiments, when items were arranged in trays as shown in FIG. 7 including baffles (760), with or without aperture(s) (765), highly uniform cooling was observed.
FIG. 8 illustrates in embodiments of the disclosure a chart of exemplary time versus temperature rate-controlled cooling achieved with an appropriately partially filled tray in a freezer system of, for example, FIG. 6 or 7. Specifically, the chart shows the measured temperature relationships of the averaged in-sample thermo-sensors (820) temperatures (top trace) and the averaged chamber thermo-sensors (840) temperatures (bottom trace). The solid line (870) provides a targeted or reference cooling-rate slope representing approximately −1°C. per minute. The dip in chamber temperatures represented by step (880) illustrates the latent heat-of-fusion of freezing samples caused by dropping the chamber temperature to about −40 to about −90° C. to promote uniform freezing of sample items. The chamber temperature excursion, preferably of limited duration, for example about 5 minutes, such as to avoid damage to sensitive sample items, is not believed to be adverse to the sample cooling-rate since near equilibration (see after about 45 minutes) of sample and chamber temperatures is apparently again achieved when freezing of sample items to a solid state is completed.
FIG. 9A illustrates a cross-sectional view of a stackable tray (900) of a rack system in embodiments of the disclosure, including one or more tray plates or sheets (910) having major and minor openings, and tray base plate or sheet (920) which is free of openings, or alternatively, the tray base sheet has openings which are offset from the major and minor openings in tray sheets (910) so that enhanced circulation can be provided but where samples cannot inadvertently be dropped or readily fall through any of the base plate openings. There is also illustrated spacer-support connector member assembly (930), vacant openings (940), a representative occupied sample opening (950) which is holding sample item (960), and non-aligned apertures (955) on the base plate (920). Also shown are optional end-baffle members (970) and (in phantom) an optional second stackable tray assembly (980) atop tray assembly (900). In further detail FIG. 9B, the spacer-support member assembly (930) can have a fastener (931), which connects, for example, raised washer (932), top plate (910), and through-rod member (933), and optional second fastener (939). The through-rod member (933) can be encased by, for example, spacer bushing member (934). The spacer-support member assembly (930) can optionally have internal wiring (935) which can provide a connection (936) for an optional “in-sample” thermo-sensors, to provide embedded wiring (937) for in-plate thermo-sensors, or to provide an optional electrical inter-connection (928) between, for example, stacked trays (938) or to connect to the external controller. The raised portion of washer (932) permits convenient stackable engagement with a foot or feet, that is the end of the spacer leg support member, of another tray assembly (980).
FIG. 10 illustrates in embodiments of the disclosure, a perspective view of a stackable tray (1000) of a rack system, including top tray plate (1010) having major and minor openings (1015 and 1017 respectively), optional middle tray plate (1020) also having major and minor openings (1015 and 1017 respectively), and base- or bottom-tray plate (1030) which can be free of openings, or alternatively, the tray base plate has “non-accommodating” openings which are preferably offset from the major and minor openings in tray plates (1010 and 1020) so that enhanced circulation can be provided but where samples cannot be inadvertently dropped or readily fall through any of the base plate openings. Thus, for example, top plate (1010) and middle plate (1020) each have a matching array of 14 (across) by 10 (up) main openings (1015) and 13 (across) by 9 (up) minor openings (1018). The lower plate (1030) can have, for example, 13 (across) by 9 (up) minor openings (1018), and preferably having no direct vertical path alignment to prevent sample fall-through. There is also illustrated spacer-support member assembly (1040) and optional end-baffle members (1050). As mentioned and illustrated in FIG. 9, the tray shown in FIG. 10 can also be stacked, electrically interconnected, or both (not shown) if desired.
FIG. 11 illustrates another stackable tray in embodiments of the present disclosure, which shows a perspective view of a stackable tray (1100) of a rack system, including top tray sheet (1110) having major and minor openings (1115 and 1118, respectively), and base sheet (1130) or bottom-tray plate which can be free of openings. Alternatively, the base sheet has vacant openings (1132) which are not co-linearly accessible from the top sheet, but are instead offset from the major and minor openings in top sheet (1110) so that enhanced circulation can be provided but where samples cannot drop- or fall-through any of the base sheet openings. Thus, for example, top plate (1110) has an array of apertures: a first set of 14 (across) by 10 (up) main- or major openings (1115) and a second set of 13 (across) by 9 (up) minor openings (1118). The base sheet (1130), for example, can have apertures (1132), such as, 13 (across) by 9 (up) openings, and preferably having no direct vertical path alignment (i.e. non-collinear) (as shown) to prevent sample fall-through. The base sheet apertures (1132) can have dimension (diameters) the same or similar to the major openings (1115) or the minor openings (1118). Alternatively, the base sheet apertures (1132) can have a dimension (e.g. diameters) different from either the major openings (1115) or the minor openings (1118). There is also illustrated end-baffle members (1150) optionally having end-apertures (1151) or openings which can be arranged to permit or achieve various useful alternative refrigerant air-flow pathways and perturbations thereof, for example: across the top of sample items, across the bottom of sample items, across the middle of sample items, or combinations thereof. As mentioned and illustrated in FIG. 9, the tray shown in FIG. 11 similarly can also be stacked, electrically interconnected, or both (not shown) if desired, for example, using optional stacking aperture(s) (1153) situated on the bottom of the end-baffle (1150) and which apertures (1153) can be adapted to receive optional stacking pin(s) (1154) or like members, situated on the top of the end-baffle (1150). The connector assembly in this embodiment can comprise, for example, two end-baffles connected to opposite ends of the tray. The connector assembly of this embodiment can also provide the abovementioned leg members by, for example, having the end-baffles extend below the base sheet, the top sheet, or both (as shown). In embodiments, one or more trays having baffles attached to both ends of the tray are preferably oriented perpendicular to the direction of the main flow path of circulating refrigerant. The main air-flow path can be readily determined, for example, using a number of thermo-sensors when the freezer chamber is vacant, that is, without a rack system or a tray present.
The present disclosure also contemplates trays which can include, as shown in FIG. 11, a plug or stopper (1156), such as rubber stoppers or like temperature insensitive materials, which can be used to selectively alter, completely or partially, the air-flow patterns through apertures of the trays, such as any of the above-mentioned major or minor apertures, and apertures optionally situated in the end baffle members. Using one or more plugs enables an operator to empirically optimize, or alternatively, customize flow patterns in the freezer system and rack system for particular needs or applications. The present disclosure also contemplates trays, which can optionally include a baffle member situated on a surface of the tray sheet, such as the optional rib-type baffle member (1158) on the top surface of top sheet (1110). The present disclosure also contemplates trays that can satisfactorily be used when upside-down or inverted, for example, placing items in the base sheet (1130) apertures (1132) and being supported by the baffle (1150) and pins (1154). In another alternative construction, the baffles (1150) can be inverted, that is pegs or pins (1154) downward and apertures (1153) upward, while the tray sheets (1110, 1130) remain oriented as shown.
The following examples serve to more fully describe the manner of using the above-described disclosure, as well as to set forth the best modes contemplated for carrying out various aspects of the disclosure. It is understood that these examples in no way serve to limit the true scope of this disclosure, but rather are presented for illustrative purposes.

COMPARATIVE EXAMPLE 1

Evaluation of Cryomed Freezer to Provide Rate-Controlled Cooling Failure of the commercial rate-controlled freezers to provide rate-controlled cooling at a desired rate of about −1° C. per minute was particularly evident when the sample load, i.e. the total amount of sample material placed in the freezer, was increased, e.g. from about 0.5 mL of sample contained in about 1 mL ampoule to about 5 to about 50 mL of sample contained in a 100 mL ampoule.
Specifically, in one exemplary test the commercial freezer, a Cryomed 2700−C., see U.S. Pat. No. 4,030,314, having a chamber volume of about 30 liters, and having fully loaded sample trays with each tray having, for example, 162 sample ampoules containing about 10 mL of sample each and total volume of about 4,863 mL (3×1,620 mL) was unable to maintain a constant or rate-controlled cooling at the desired rate of about −1° C. per minute. Instead, an irregular or erratic cooling-rate and temperatures within the chamber and within individual samples were observed. For example, when the chamber temperature was apparently at −90° C., samples situated to the periphery of a tray were colder or frozen whereas samples situated towards the center of the tray, such as forming a circle having about a 10 ampoule or aperture diameter, were unfrozen, see for example the above mentioned warm-zone (450) of FIG. 4. The observed non-uniform cooling-rate and temperature differential among similarly situated samples, particularly the existence of unfrozen samples at an apparent chamber temperature of −90° C. was unacceptable and undesirable for many preservation applications.
Additional experimental trials directed at achieving uniform cooling-rate and comparable freeze temperatures among samples, for example, by increasing the cooling-rate, such as to a rapid cooling rate regime of about 5 to 10 ° C. per minute did not overcome the above-mentioned differentials nor the existence of unacceptable unfrozen liquid samples.

COMPARATIVE EXAMPLE 2

Evaluation of Cryomed Freezer to Provide Rate-Controlled Cooling Comparative Example 1 was repeated with the exception that the sample trays were filled with ampoules as shown in FIG. 5, having main openings filled with ampoules and minor openings vacant for a total of 90 openings per tray filled with ampoules of the 162 available openings. A cooling rate of about −1°C. per minute produced similar non-uniform cooling results as in Comparative Example 1, for example, some ampoules were observed and measured to have unfrozen liquid sample content, i.e. temperatures of greater than about 0° C. when the chamber temperature was measured at −65° C., while sample nearest the coolant source were frozen solid, e.g., the right edge of the tray.

COMPARATIVE EXAMPLE 3

Comparative Example 2 was repeated with the exception that a commercially available Kryo 750-30 freezer was used, having two rectangular trays that could accommodate 9×16 or 144 ampoules per tray for a total capacity of 288 ampoules. The trays were fully loaded and were without unoccupied holes or apertures, and then cooled with the result that a pronounced gradient cooling pattern was observed where the ampoules nearest the cooling source froze solid while ampoules furthest away from the cooling source were unfrozen.

EXAMPLE 4

Evaluation of Cryomed Freezer to Provide Rate-Controlled Cooling Comparative Example 2 was repeated with the exception that the sample trays were filled with ampoules as shown in FIG. 6 having alternate or every-other main openings filled with an ampoule and all minor openings vacant for a total of 45 openings per tray filled with ampoules of the 162 available openings, such that no filled opening, that is an ampoule occupied opening, has a nearest-neighbor opening that is filled or occupied with an ampoule. A cooling rate of about −1° C. per minute produced nearly uniform cooling and freezing results.

EXAMPLE 5

Evaluation of Kryosave Freezer to Provide Rate-Controlled Cooling Example 4 was repeated with the exception that the freezer was a Kryosave 750-30 having a laminar flow pattern characteristic. A cooling-rate program was used which automatically compensates for the total number of sample ampoules in the chamber and their collective impact on the total heat of fusion sample load. A cooling rate of about −1° C. per minute produced nearly uniform cooling and freezing results.

EXAMPLE 6

Evaluation of ThermoForma Freezer to Provide Rate-Controlled Cooling Example 4 was repeated with the exception that the sample trays had a slightly different configuration and capacity. The trays were filled with ampoules as shown in FIG. 7 and a ThermoForma Model 7454 freezer, see the abovementioned U.S. Pat. No. 6,044,648, was used having a larger fan, a higher capacity fan motor capable of providing greater convection, a larger chamber, a heat transfer coil based on liquid nitrogen “spray,” and capable of accomplishing the following instruction “hold chamber at temperature X until sample temperature is Y”.
In preliminary experimental trials with this freezer in several configurations, such as using one, two, or three trays, it was apparent from the sample temperature data and the cooling-rate measurements that the cooling pattern within in the chamber can be markedly affected by the rack and tray configuration. This observation prompted further modification of the apparatus to include one or more baffles and explore the influence that a baffle had on temperature and cooling rate, specifically whether early cooling of sample(s) near the refrigerant source and late cooling of sample(s) away from the refrigerant source could be prevented or attenuated.

EXAMPLE 7

Evaluation of ThermoForma Freezer with Baffling to Provide Improved Rate-Controlled Cooling Example 6 was repeated with the exception that the sample trays were of slightly different design and used different sample configuration and sample loading. The sample trays were modified to include the optional baffles on both ends to obtain improved convection and refrigerant air-flow along the spaced channels between samples and within the trays. The sample configuration used was as shown in FIG. 7. This configuration permitted a sample loading of 90 sample ampoules per tray and total sample load of 270 ampoules in 3 trays. The observed temperature and cooling rate data is shown in FIG. 8. The observed temperature and cooling-rate data suggested that the end-baffles did indeed prevent or minimize undesirable premature near-sample cooling and diminished late far-sample cooling, wherein “near-sample” and “far-sample” refer to proximity of the sample item to the cooling coil and fan. Although not wanting to be limited by theory, the improved cooling results are believed to be attributable to the increased turbulent circulation of the refrigerant-air mixture within the chamber and around the sample items.

EXAMPLE 8

Evaluation of ThermoForma Freezer with Baffling to Provide Improved Rate-Controlled Cooling The results of Comparative Examples 1-3 suggested that optimal high sample volume cooling might be achieved by selecting a freezer, tray, and rack combinations which possessed the greatest volume capacity, and the greatest coolant flow and dispersion. For example, a freezer system having a large volume freezer chamber which could accommodate more than one sample tray, rack or tray assembly, and which could hold ampoules with volumes of from about 5 to about 100 mL. Better, that is more efficient and higher capacity, liquid nitrogen injection and higher refrigerant circulation capacity, for example, a larger or faster fan might also might significantly contribute to achieving high sample volume cooling. The optimal baffle configuration was found to depend on, in this and other experiments, for example, freezer size, rack size, tray size, ampoule size, sample volume size, and like considerations.
Thus, Example 7 was repeated with the exception that the sample trays were the design indicated in FIG. 9, that is, stackable trays and without the above-mentioned rack frame, and optionally having integral thermocouples and wiring, with the result that satisfactory constant rate cooling of a high total volume of samples was achieved.
From various studies of freezer models, rack configurations, tray designs, baffle placements, and like design aspects, it was evident such design aspects can be important in influencing the success of large-volume constant-rate cooling of samples. Thus, although not desired to limited by theory, it is believed that to achieve optimal high volume cooling there preferably should be: 1) maximum intra-tray air circulation or cooling, that is for example, holes or apertures situated between samples; 2) maximum inter-tray circulation, that is holes or apertures in trays which permit high coolant or air circulation between trays; and 3) a rectangularly shaped freezer chamber having rectangularly shaped trays fitted with baffles. The optimal baffle configuration was found to be dependent upon a number of factors, for example, the freezer, such as the manufacturer and the freezer's chamber geometry, rack geometry, tray geometry, tray aperture size and placement, ampoule size, sample loading, sample size or volume in the ampoule, and like factors.
All publications, patents, and patent documents are incorporated by reference herein in their entirety, as though individually incorporated by reference. The disclosure has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the disclosure. The above specification, examples and data provide a complete description of the manufacture and use of the composition of the invention. Since many embodiments of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended.

Claims

1. A freezer system comprising:

a freezer to cool items;

a thermo-sensor to monitor temperature within the freezer;

at least one tray having apertures to hold the items within the chamber of the freezer and to provide uniform cooling of the items;

a programmable controller to receive and process signals from the thermo-sensor and to control the temperature of the freezer and the cooling-rate of the items; and

a baffle within the freezer.

2. The freezer system of claim 1 wherein the baffle is part of a tray.

3. The freezer system of claim 1 wherein the baffle is situated so that the baffle disturbs laminar refrigerant flow and causes turbulent refrigerant flow.

4. The freezer system of claim 1 wherein the at least one tray comprises from two to ten trays.

5. The freezer system of claim 1 wherein at least one thermo-sensor monitors the temperature of one or more locations on a tray, monitors the temperature of one or more of the items in the tray, monitors the temperature within the freezer, or combinations thereof.

6. The freezer system of claim 1 wherein at least one thermo-sensor is situated within the chamber of the freezer and away from the freezer's refrigerant source.

7. The freezer system of claim 1 wherein the items comprise from 10 to about 500 sample items.

8. The freezer system of claim 1 wherein the programmable controller signals a cooling-rate of from about −0.1 to about −5° C. per minute.

9. The freezer system of claim 1 wherein the programmable controller signals a cooling-rate of about −1° C. per minute.

10. The freezer system of claim 1 wherein programmable controller signals a cooling-rate of from about −0.1 to about −1° C. per minute over a temperature range of from about 40° C. to about −100° C.

11. A freezer system comprising:

a freezer comprising a housing having a cooling chamber therein;

a rack system comprising one or more trays in the chamber, the trays being adapted to receive and hold items for cooling;

at least one baffle associated with the rack system or chamber; and

a temperature controller adapted to measure temperatures within the freezer, adapted to control the temperature, and adapted to provide a constant cooling-rate for the items in response to the measured temperatures.

12. The freezer system of claim 11 wherein the temperature controller comprises a plurality of thermo-sensors adapted to measure the temperature of the chamber, the rack system, and items in the rack system.

13. The freezer system of claim 11 wherein the freezer further comprises a liquid refrigerant source, a valve and solenoid adapted to regulate the liquid refrigerant admitted to the housing, a coil adapted to vaporize the liquid refrigerant, and a fan adapted to circulate the vaporized refrigerant through the chamber.

14. The freezer system of claim 13 wherein the temperature controller is adapted to control the solenoid and the fan.

15. The freezer system of claim 11 wherein each tray has at least one baffle attached to or integral therewith.

16. The freezer system of claim 11 wherein each tray is adapted to stackably engage another tray.

17. The freezer system of claim 11 wherein the rack system comprises a frame adapted to slidably receive the trays.

18. The freezer system of claim 11 wherein the freezer system provides a relatively constant cooling rate of about −1 centigrade per minute over from about 35° C. to about −90° C.

19. The freezer system of claim 11 wherein the freezer system provides a temperature variation within an item of less than from about 0.01 to about 0.5° C.

20. The freezer system of claim 11 wherein the freezer system provides a temperature variation between or among two or more items of less than from about 0.1 to about 5.0° C.

21. The freezer system of claim 11 wherein the freezer system provides a temperature variation between any two locations in the rack system of less than-from about 0.1 to about 5.0° C.

22. The freezer system of claim 11 wherein the baffle perturbs laminar air-flow patterns which are present in the chamber in the absence of the baffle.

23. The freezer system of claim 11 wherein each tray has at least two baffles attached to or integral with.

24. The freezer system of claim 11 wherein the baffle is attached to or integral with an interior wall of the chamber.

25. The freezer system of claim 11 wherein the rack system is adapted to slidably engage the chamber.

26. The freezer system of claim 11 wherein the rack system is adapted to provide an electrical connection to the temperature controller when slidably engaged in the chamber.

27. The freezer system of claim 11 wherein the rack system is adapted to slidably engage one or more trays inserted into the rack system.

28. The freezer system of claim 11 wherein the rack system is adapted to provide an electrical connection between the temperature controller and one or more trays when inserted into the rack system.

29. The freezer system of claim 11 wherein each tray has a plurality of apertures there through, the apertures being adapted to receive and hold items, and adapted to permit cross-flow refrigerant circulation if unoccupied.

30. A tray comprising:

a base sheet adapted to support items, the base sheet having an optional array of apertures there through, the base sheet apertures when present being adapted to be vacant to permit cross-flow refrigerant circulation in a freezer;

a top sheet having an array of major and minor apertures there through, the top sheet array of major and minor apertures being adapted to be non-collinear with the optional array of apertures of the bottom sheet, the each major aperture being adapted to either hold a item or to be vacant and to permit cross-flow circulation of circulating refrigerant in a freezer, the array of minor apertures being adapted to be vacant and to permit cross-flow refrigerant circulation in a freezer; and

a connector adapted to fixably join together and space apart the adjacent sheets of a tray from each other, and optionally adapted to permit two or more trays to be stacked together.

31. The tray of claim 30 further comprising a baffle connected to tray or integral with the tray.

32. The tray of claim 31 wherein the baffle is connected to at least one of the sheets.

33. The tray of claim 32 wherein two baffles are connected to opposite ends of the tray, the ends being oriented perpendicular to the main flow path of circulating refrigerant when the tray is placed in a freezer.

34. The tray of claim 30 wherein the connector comprises a baffle, the baffle optionally having apertures there through, the baffle apertures being adapted to selectively permit refrigerant circulation through the baffle or to be plugged.

35. The tray of claim 34 wherein the connector further comprises a leg member adapted to elevate the base sheet to afford an air-way beneath and traversing the base sheet and optionally adapted to permit tray stacking.

36. The tray of claim 30 wherein the connector comprises two baffles connected to opposite ends of the tray, the ends being oriented perpendicular to the main flow path of circulating refrigerant when the tray is placed in a freezer.

37. The tray of claim 36 wherein the connector further comprises a peg member adapted to elevate the base sheet to afford an air-way beneath and traversing the base sheet and optionally adapted to permit tray stacking.

38. The tray of claim 30 wherein the connector comprises a connector rod, an optional spacer, an optional nesting washer, and a fastener.

39. The tray of claim 38 wherein the optional nesting washer is adapted to stack and electrically interconnect two or more trays.

40. The tray of claim 30 wherein the connector is further adapted to permit electrical interconnection of the tray to another tray and to permit electrical connection of the resulting tray to a temperature measurement system.

41. The tray of claim 30 further comprising a middle sheet interposed between the top sheet and bottom sheet, the middle sheet having an array of major and minor apertures there through substantially identical and co-linear to the top sheet apertures.

42. The tray of claim 30 further comprising a baffle member attached to a surface of a tray sheet.

43. A rack system comprising a tray of claim 30 adapted to receive and hold items.

44. The rack system of claim 43 comprising 1 to 10 trays.

45. The rack system of claim 43 wherein the connector comprises a baffle, the baffle optionally having apertures there through.

46. The rack system of claim 43 further comprising a baffle member on a surface of a tray sheet.

47. The rack system of claim 43 wherein the tray holds from 60 to 150 items.

48. The rack system of claim 43 wherein the item comprises an ampoule having a sample volume of from about 5 to about 150 mL.

49. The rack system of claim 43 wherein the tray has a total ampoule sample volume capacity of from about 1,000 to about 15,000 mL.

50. The rack system of claim 43 wherein the size of the items are the same or different.

51. The rack system of claim 43 wherein the item comprises an ampoule containing a biological material.

52. The rack system of claim 43 wherein the item comprises an ampoule containing living cells.

53. The rack system of claim 43 wherein the item comprises an ampoule containing antibodies.

54. The rack system of claim 43 further comprising a shaker adapted to shake the rack and agitate the items in the tray or trays.

55. A method of cooling with a freezer system, the freezer system comprising:

a freezer adapted to cool items;

a plurality of thermo-sensors adapted to monitor the temperature at points within the freezer;

a programmable controller adapted to receive and process signals from the thermo-sensors and further adapted to send signals to control the temperature and the cooling-rate of the items in the freezer; and

a tray comprising:

a top sheet having an array of major and minor apertures there through, the top sheet array of major and minor apertures being adapted to be non-collinear with the optional array of apertures of the bottom sheet, the each major aperture being adapted to either hold a item or to be vacant and to permit cross-flow circulation of circulating refrigerant in a freezer, the array of minor apertures being adapted to be vacant and to permit cross-flow refrigerant circulation in a freezer;

a connector adapted to fixably join together and space apart the adjacent sheets of a tray from each other, and optionally adapted to permit two or more trays to be stacked together; and

optionally a baffle connected to an end of the tray;

the method comprising:

arranging a plurality of items in a pattern in the tray;

placing the tray in the freezer's chamber and sealing the chamber;

circulating the air and a gaseous refrigerant through the chamber and in contact with the items;

monitoring the temperature at points in the chamber with the thermo-sensors; and

controlling the temperature and the cooling-rate of the items in the freezer with the programmable controller.

56. The method of claim 55 wherein the pattern of items comprises having an item occupy an aperture and having adjacent apertures vacant.

57. The method of claim 55 wherein the pattern of items comprises having an item occupy the major apertures and having minor apertures vacant.

58. The method of claim 55 wherein the cooling-rate is about −1° C. per minute over a temperature range of about 40 to −100° C.

59. The method of claim 55 wherein the method provides uniform temperatures within individual items and uniform temperatures among items.

60. The method of claim 55 wherein the method provides rate-controlled cooling within individual samples and among items.

61. The method of claim 55 wherein the method provides a narrow temperature differential among items of about 0.5° C.

62. The method of claim 55 wherein the method provides a temperature differential between the chamber and the items in the chamber of about 5.0° C. to about 50° C. for less than about 10 to about 20 minutes.

63. The method of claim 55 wherein the controller controls the temperature and cooling-rate of items by regulating the amount refrigerant admitted to the chamber and the circulation rate of the air and refrigerant in the chamber.

64. The freezer system of claim 1 wherein the aperture includes a valve optionally connected to and regulated by the programmable controller.

65. The freezer system of claim 1 wherein the baffle is moveable by a mover and the baffle's movement is optionally regulated by the programmable controller.