CA1058522A - Method of and apparatus for the deep freezing of biological substances - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A system for the deep freezing of biological sub-stances provides an input representing the temperature-time curve required at the outer wall of a receptacle containing biological substances to be deep-frozen while a sensor measures the actual temperature at this wall and controls the cooling applied to the receptacle to conform the cooling at the receptacle wall to the precalculated temperature-time curve. This permits the necessary temperature gradient to be applied to the biological substance for maximum cell survival without any dead time necessitated by the use of sensors within the biological substance itself.

Description

~s~s~z Field of the Invention The presen~ invention relates ~o a method o and to an appara~us ~or ~he deep-~reezing of biological substances ln re-specti~re receptacl~s and, more particularly, to the de~p-reez-ing of biological substanees which have b~en introduc2d into s~-c~lled biorecep~acles and are sealed ~herein pr~or ~o being deep-frozen by means o~ a ooolant or refrigerant such as liq~efied nitrogen.

In cryogeni-~ processes for ~he pre~ervation of b~ologi-cal substances such as blood, blood components, cell ~usp~n-sions and cell tissue~, the majnr problem resides in ~voiding irreversible cell d~mage which can ~esult during the ~reezing process and the subsequent th~wing process, or the minimizir~g of such damage .
It has been proposed heretofore to limit th~ cell damage of biological substances o~ ~he ch~racter described by ~he addition of a cryophylactic protective additive or agent which serves to protect the cells against the effec~s of :Ereezi~g 20 and thawing and which is mixed with the cell su~pension or other biological substance. Such protect~ve agents increase ~he sur~ival rate of ~he frozen cell materials.
Pro~ec~ive add~ res such as glycerin have been used here~ofore, especially ~or the protection o blo~d against t~
effects of the deep-freezing process, and must be washed from the preservPd biological substances after thawing because ~hey can adversely afect the human organismO ~C:onsiderable research ~s~szz has gone in~o ~he developmen'c of biologically innocuous pro-tecti~7e addi~ives and, when such are employed, the survival rate can be :incre~sed.
Investigations have shown tha~ an lmportant factor in avoiding the decompositioa or destruction o:E ~e cells is the tempera~ure gradient with which the cells are ro2en. In other words, there are prede~e~minable cell-speciic ~ime-dependen~ temperature gradients ~t which cellular m~-terial, i~,eO the biolo~ical substances described above, ~an be 10 frozen to ob~ain a survi~al ra~e o about 98%. This latter percentage has been found ~o be a reasonable level for mos~
cryogen~c deep-freezing processes and, when reference i5 made herein ~o ~ime-dependent cell-specific temperatur~ gradients, i~ will be unders~ood that such gradients are intended as will ensure a cell survival rate of about 98% following deep ~reez-ing and thawing.
When ~he speed of the freez~ng process lies ben~ath this ~er~pera~ure gradient, the concen~ra~ion of the extra-cellular liquid is increased during the reezing process by 20 the ~reezing out of water therefrom. This results ln an in-crease in the osmotic pressur~ be~ween the inner-cell and outer-cell media. Furthermore, dur-lng the fre~zing process water is wi~hdrawn from the cells themselves and this results in a concen~ration increase in the intracellular solution as well. miS can gi~e rise to den~turation of ~he proteins in the cell interiorsO While the ef~ects of such processes can be minim~ed by an increase in the sp~ed o~ ~he freezing pro~
cessS t~ere ~e~ertheless is a tendency at both excessively high speeds and low speeds ~o produce intercellular ice which, in any case~ breaks down the cell walls and membranes.

~S~5zz Qf course, the amount and type of protective agent will also influence the desired temperature gradien~ o~ the freezing process. For example, when mix~ures of erythrocytes with glyc erin in high concentrations o about 50% are subjec~ed to deep-~reezing at a temperature gradîent of about ~ K/min (8 de-grees Kel~rin or Cen~igrade per minu~e~, high survival rates of the blsod cells are noted. For unprotected erythrocytes, the optimum temperature gradient is about 50~0 E~/min and even a~
this op~lmum, the maximum survival rate o~ the c211s iS found 10 to be only about ~0~0.
Known processes for the deep-cooliIlg preservation o~
biological substances, which can be contained in so-called b~orecep~acles, eithe~ maintain the biological recep~acle in a liquid nitrogen bath for a predetermined time period, some~imes wi~h shaking in order to ensur~ effective mixture o the bio-logical substance wi~h ~he protective agent, or spray ~he bioreceptacle with liquid nitrogen while monitoring the tem-pera~ure within the interior of the recep~acle.
The recept~cle which can be used in the prior~art ~0 systems and in the invention described ~elow can be any syn-the~ic resin sack or other conta~ner con~entionally used to receive mixtures of blood and protective agents or other bio-logical substances admixed with protec~i~e agentsO
By the technique descr~bed above, the freeging process canno~ be accur~tely maintained at a predetermined cell~spe-cific ~empera ture gradientO
The immersion process, which can be limited only as to t~me, does not permit Yariation in the temperature gradien~
under such controls as ~o main~ain a predetermined cell-specific 30 ~emperature gradient and the optimum temperature gradient ~or any specif cell can, at best, only be approach~d.

~s~

me~sp~ay process permits a monitoring o the change of temperature with time by means of a thermoelemen~ in the interior of ~he bioreceptacle~ but has the diæadvantage khat there ls a large ime lag in the con~rol process, i.e. the reaction time between a change in the supply o the coolant and the re~ulting change in the ~empera~re in ~he ~n~erior of ~he bioreceptacle is considerable. This, ~oo, pre~en~s an accurate control of the ~emperature gradien~O

Qb'ects o~ the Invention Xt is the principal ob~ect o the presen~ invention to provide a process and an apparatus for ~he deep~freezing of biological ~ubs~ances contained in b~oreceptacles, in which during the freezing process a cell-specific temperature gradi-ent op~imum for the specific biological substance can be maintained with high precision and high reproducibili~y.
It is another object of ~he ~nventlon ~o provide a sys~em for the deep-~reezing o biological substances, such as those mentioned abo~e, with or without protect~ve agents, whereby the aforementioned disad~antages are avoided.

Summary o~ ~he Invention These ob~ects are attained, in accordance with the present invention, in a system (proces~ and/or appara~us) whereby the temperature of the outer wall of ~he bioreceptacle ~s controlled as a f~n~tion of time to conform ~o the tempera-ture-t~me curve which ~s calculated to respond to the optiswm ~.emperature gradient for any speclfic biological s~bstance at the outer wall of the bioxeceptacleO
In o~her words, according to the in~ention~ when a predete~mined temperature gradient is to be maintained during the freezing process ~o ensure approximately 98% survival ~s~

rate of ~he cells of this biological substance upon deep~
-freezing, the temperature-time curve a~ the outer wall o ~he biorecep~acle necessary to maintain ~his predetermined temper-a~ure gradient is firs~ calcula~ed and ~he deep-freezing process is con~rolled so that the tempe~ature at the outer wall of the bioreceptacle varias as a 1mction o~ time ~o correspond ~o ~his calcula~ed tempera~ure- ~ime curve .
~ y precalcula~ing the temperature- time cun7e or the ou~cer wall of ~he biorecep~acle, which yields the des~red tem-10 perature gradien~ for ~he biological su~s~ance in the interis:rof the bioreceptacle, and by conforming the change in tempera-ture at ~he outer wall of the bioreceptacle wlth time to correspond to this calculated temperature-time curve, it is possible in accor~nce with the in~ention to carry out the freezing process of any given biological substance with the desired temperature gradien~ without concern or dead ~ime, ~hermal inertia or lag time in a control processO
An important charac~eristic of the inven~ion is tha~
it permits a thermoelement in the interior of the biorecep-2û tacle to be completely dispensed wi~h and i~ alæo eliminatesthe effects of long reaction times resul~ing in dela~s in the change in ~h~ temperature within the bioreceptacle.
Becallse of the mathematical solution which is used ~o calculate the ~emperature within ~he biorec~ptacle, all meas-urements of the temperature wi~hin the interior of ~he bio-logical substance in ~he bioreceptacle can be eliminated~. ~he calculation, of course, takes into consideration the thick-ness of ~he wall of the bioreceptacle, the coefficient of thermal conduction thereo, its heat capacity and the heat-30 -transer coefficien~ between the cooling fluid and the receptacle w~ll and between the receptacle wall and the ~OSB5ZZ

biological substances as well as ~he thermal characteris~ics of ~he liquid layers and the interfacial ~hermal characteristics between the receptacle and the fluidso According to one aspec~ of the inven~ion7 the reeæing procQss is controlled ~o correspond ~o th~ c~lculated ~emp~ra-ture-time curve when the bioreceptacle is sprayed with a lique-fied coolan~ especially ni~rogen, and ~he supply o~ ~he cooling medium per unlt ~ime is regulated in dependence ~pon the ~empe~
ature measured at the out~r ~all of the bioreceptacle. A lag in con~rol~ o~ ~he type which occurs when the measurement Qf ~he temperature takes place in the inter~or of the receptacle, is excluded. The desired ~empera~ure gradien~ can be accurat~ly maintailledn According to ano~her aspec~ or ~eature of the invention, the bioreceptacle can be electrically heated externally during the ~reezing process so that the deslred change in temperature with time is maintained at the ou~ex wall of the bioreceptacle which is sub~ec~ed to deep-freeze cooling by~ for e~ample, the spray-cooling t~chni~ue mentioned above or by immersion cooling, 0~ course, in this case~ ~he heat abstracted by the coolant must exceed the heat del~vered by the electrical heating meansO
I~ h~s been found that the electrical hea~ing technique permits a hlghly exact contro~ of the temperature on the external sur-face of the recep~acle and hence main~aines a predetermined temperature gradient.
It has been ~ound to be highly advan~ageous, in the dee~
freeæing of cells and biological substances which require rela-~ively low temperature gradients of ~ew K/min, ~o avoid cell damage, for e~ample for the ~reezing of corpuscular blood com~
ponents such as thrombocy~es or lymphocytes, to provide ~h~

~195~52;2 freezing apparatus with a pair of pla~ces of low ~hermal conduc-tivity and to dispose the bioreceptacle between these plates.
These plates can be composed of synthe~ic resin and have wall thicknesses which are ~alcula~ed, iLn dependence upon the tem-perature-time curve for the outer wall of t:he recep~acle, to provide the desired temperature as a function of ~cime a~ this outer wall. The assembly of the low-~hermal-conduc~iYity plates and the bioreceptacle can ~hen be immersed in a lique-~ied gas~ e.g. liqu~fied nitrogen~ forming the coolant bathO
The bior~ceptacle can also be hea~ed, preferably elec-trically, along its external sur~ace even in ~he immersion, to maintain the temperature function of ~ime at the ex~rnal sur-face of the receptacle in conformity with ~he calculated tem-perature- time curve .
In this case, the desired temperature grad~ent can be provided as a first appro~ ion by the controll of the wall ~hickness of the plates o low-thermal-conductivity material and can be corr~cted by heating the outer wall of ~he biore-ceptacle in response to an actual measurement of the tempera~
ture a~ this wall, the measured temper~ture being compared with ~he calculated tempera~ure at any ins~ant ln time of the temper-ature-time curve to produce an error signal which controls the heating.
The low-heat-conductivity pla~es ~hus provide a ooa~se control o the freezing speed while the heating operation main-tains the fine control thereof.
An ~pparatus for carrying ou~ the process o~ the pr~sent invention, using the spray-cooling technique, advantageously comprises a cooling channel disposed in a sterile chamber and 30 pxovided wi~h feed means for supplying the liquefi~d coolan~

~ ~ ~ 8 5 ~ ~
and a control or regula~ing unit ~con~roller) wuch that the liquefied coolant spray de~ice is connected ~o the source o liquefied coolant while the latter is connected, in ~urn, to the con~roller.
Ad~antageously, the cooling channel can be dispos~d vertically and can be pro~ided, along its opposite 1anks, with copper pipes whose noæzles are trained toward one another and agains~c the bioreceptacle which is introduced between ~he copper pipes so ~ha~ the lique~ied coolant is spr~yed direc~ly on~o ~he surface of the bioreceptacleO
It is especially advanta~eous, in accordance with the invention, to provide a thermal elcment (temperature sensor) within th~ cooling channel such that it lies in direct contac~
with the outer surface of the bioreceptacle and is connecl:ed to the controller for opera~ing same~
Based upon the geometry and materials of the recep~a~le and of the cooling channel, the temperature-time curve for the outer wall of the bioreceptacle, based upon the desired ~empera-ture gradien~ of ~he biolo~ical substance therein, can be readi-i 20 ly calculated and can serve as a se~ point value for ~he con-troller, being compared, at any instant, wi~h the measured temperature to produce a signal which is employed to control ~he supply device for the lique~ied coolant.
This not only has ~he advantage that it can carry out the deep-~reezing under fully sterile conditions, WithOtlt con-tact o the biol.ogical substance with the temperature sensor or any extranecus element, bu~ also avoids the problem of thermal iner~ia or dead ~ime in the control process.
According to another ~eature of the in~en~ion, a holder for the biorecep~acle is pro~ided within the cooling channel between the spray systems and is adapted to receive the 3S2z bioreceptacle sueh ~a~ the lat:ter lies in contac~ w~h ~he suraces o the holder. A surace o the holder ln con~ac~
with the bioreceptacle can be provided wlth a temperature--sensing elemen~ described above while the outer ~urface of ~h~ holder pla~es can be provided with electrical hea~cing devices for the pusposes descrlbed previouslyJ
The holder plates can be sheet me~al elements con~oured to rece~Te the receptacle and can be urged agains~ the la~t0r by spring and/or lever devices which can be used to spread ~:he plates when t~e reccptacle i~ recelved and ~o ~irmly hold the pla~es in surface-to-surface contact with the outer walls of the receptacle when the latter is subjected to deep-freezing.
The hea~ing means can be electrical heating coil5 em-bedded in s~licone rubber ~nd applied to the external surfaces of the pla~es. The amount of heating genera~ed per unit area and the amount of cooling applied by tha spray nozzles per unit area of the plates can be regulated by the controller in accordance with the ~emperat~re measured at the outer ~all o~
the biorecep~acle~
The supply deviee ~or the coolant can, according to s~ill another eature of the invention, include, besides the vessel containing the liquefied coolant7 also a vessel ~or ~he gaseous cs)oling medium such ~hat the liquefied~coolan~
vessel is connected to the gaseous-coolant vessel through the controllerO The interior of ~he l~quefied-coolant re!ceptacle can thus be maintained at a oonstant supera~mosph~ric pressure.
When, before ~he liquefied coolan~ is withdrawin, ~he liquid level ~alls below a prede~ermined height in ~he lique-~fied-coolant re~eptacle~ che oontroller is triggered by ~he pressure drop and feeds gaseous ~edium from the other recepta-cle into tlle liqueied-coolant receptacle to maintain the necessary superatmospheric pressure th~reinO

_ g _ '~S~ 2 ~ apparatus ~or carrying ou~ the process aceord-lng to the immerslon technique comprises a con~ainer for the llque-fied coolant, an immer~icn device and, advantageously, a holder which is suspended from the immersion dev~ce and which includes a pair of plates of low-thermal-conduc~i~lty material between which the bioreceptacle can be dispos~d~ The apparatu~ also includes a con~roller which respo~ds ~o a ~hermal elemen~ in contac~ wi~h the ou~er wall o~ ~he bioreceptacle and a heating device operated by the con~ro~ler. Th~ th~rmal element or ~emp~rature sensor is thus preferably mounted on the inner ~urface of a metal plate be~ween two of which ~he biorecep~acle is receivedO

Brief Des criPtion of ~he Drawin~
~ he above and other objects, features and advan~ages of the presen~ in~Te~tion will become more readily apparent from ~he ~ollowing description, reerence being made ~o the accompa-nying drawing in which:
FIG. 1 is a vertical section through a deep-freezing chamber according ~o the invention, shown in diagrammatic form, and illustrating other por~ions of the apparatus according ~o one embodiment o the inven~ion schematically;
FIG. 2 is a view similar ~o FIG. 1 bu~ illustra~ing ano~her embodiment of the invention;
FI&~ 3 is a block dlagram showing ~ control system for the purpose of ~he presen~ invention;
FIGo 4 is a graph of ~he tempera~ure (ord~nate) versus the cool-down time (abscissa) demons~rating the in~ention; and FIG~ 5 is a series o~ graphs in which the variation in temperature of a frozen suspension at a distan~e within t~e suspensi~n from ~he coolin~ surface (abscissa) is plo~ed as a ~85~Z

~unction of the applied temperature at the surface of the receptacle (ordinate).
Specific Description The embodiment of FIG. 1 uses the spray technique for the deep-freezing of biological substances of the type described while the embodiment of ~IG. 2 utilizes the immersion technique. Both embodiments can make use of a controller of the type shown in FIG. 3.
Throughout this speci~ication, when reference is made to biological substances it is intended to include therein blood, blood components, cell suspensions and cell tissues which may or may not be admixed with protective agents.
In FIG. 1, the sterile hermetically sealed chamber 1 is provided with a deep-freezin~ device 2 including a vertical cooling channel 3 having along its opposite interior walls a spray system 4 for a liquefied coolant, e.g. nitrogen. The spray system can comprise vertical copper pipes having nozzles which train the sprays of liquid nitrogen against the bioreceptacle 6 containing the biological substance, preferably in admixture with the protective agent, and packed and sealed.
Between the spray nozzles, there is provided a holder 5 for the bioreceptacle 6, the holder having external contours conforming to those of the bioreceptacle and preferably being clamped thereagainst by spring or le~er means not shown.
The pla~es of ~hei-holder can be of shee~ metal and are pro-vided along their external sur~aces, i.e. their surfaces turned away from ~he bioreceptacle, with hea~lng coils 7 embedded in layers of silicone rubber. These heating coils permit the heating of the bioreceptacle ~.
On the surace of the holder 5 contacting ~he outer wall of the bioreceptacle 6, there is provided a tempera-ture-sensing element 8 which preferably bear~ against the b~oreceptacle 6 to ensure a finm contact therewlth. ~his tempera~ure sensor maasures the ~emperatur2 on the ~xterior wall of the bioreceptacl2 6 and is connected to a con~roller 9 through which ~he heating coils 7 are energized and which also operates a valve 10 supplying the nozzle systems 4 with the liqueied coolant (liquid nitrogen) from a supply device 11~
Thus ~he amount of liquef~ed coolant supplied per unit time and the heating v~a coils 7 per unit ti~le are regulated by the con~roller 9 in response ~o the thermoelement 8 to pro~
vide a tempera~ure at the outer wall of the receptacle 6 which is a function of ~ime and con~orms to the precalculated temper-atu~e-time curve described aboveO
In order to ensure a constant flow of the liquefied coolant via th~ valve 10 to the nozzles 4, the supply unit 11 is provided with a receptacle 12 ~or the liquefied coolant and means connecting thls recep~acle 12 through the con-troller 9 to a bottle 13 supplying the gaseous coolant a~ an adjustable supera~mospher~c pressure~ Should the pressure fall ln ~he line eeding the noz~les ~ gas is fed rom bottle 13 to the receptacle 120 ~ll058~

FI(~. 2 shows an immersion deep-~reezing system in which a container 20 has a bath o~ the liquefied coolant and is pro-vided at 21 with an immersior device or lowering the biorecep-tacle into this bath.
~ ccording to the inventinn~ this immer~ion device 20 i~
designed to control the depth 1;o which ll:he recep~acle is low~
ered in~o the bath. The immersion de~7ice 21 carries a holder 22 in which the biorecep~acle 28 can be received, the holder 22 being suspended ~rom ~his immersion device. The holder il:self ~0 can be adjus~able so as to c~}mp the bioreceptacle be~ween ~che parts ~:hereof~, 8~g~ via the spring or lever means mentioned previously.
me hvlder 22 rece~ves a pair of me~al plates 25 which rest directly aga~nst the out~r walls of the bioreceptacle ~8 and can be conformed geometrically to them. Along ~he outer suraces of these metal plates, ~here arc provided synthetic-resin plates 23 o~ low thermal conductiv-lt~ which are engaged by the holder 22 only at ~he upper and lower ends. Since the holder 22 can be o~ adjustable siza, it c~n receive plates 23 20 of diff~rent thidcness, the thickness o ~he plates correspond~
ing to t:he desired temperature grad~ert to be maintaine~ in the manner described previously.
For the fine control of the freezing process, as in the system of FIGo 1~ the sur~ace of the metal plates 25 turned away ~rom ~he bioreceptacle 28 can be provided wi~h heating coils 24 embedded in silicone rubber while the surface turned toward and con~ac~ing the bioreceptacle 28 carries a ~empera-- ture- sensing element 26 which i~ connecte~ ~o the controller 27 opcrating the heating element 240 The metal plates 25 serve not only as carriers for the temperature-sensing element 26 and the heating device 24 but also impart a flat predetermined uniform configuration to the synthetic-resin sack containing the biological substances and thus serve to homogenize the heat transfer over the broad surfaces of the bioreceptacle.
As can be seen from FIG. 3, the control or regulator system 9 or 27 can include a memory 30 in which the tempera-ture-time curve is recorded upon calculation as described above, this memory supplying one input to a comparator 31 whose other input is supplies by a temperature sensor 32 which can represent the sensor 8 or 26 of FIGS. 1 and 2. A
different signal from the input of the comparator 31 can be applied to the heater 33, e.g. the electrical heater 7 or 24 of FIGS. 1 and 2, or to the deep-freezing spray control unit 34 which can be the valve 10 of FIG. 1.
By way of example, there is shown in FIG. 4 a graph of temperature-time curve as calculated for blood and the corresponding measured values as obtained by a test probe in the interior of the bioreceptacle. The latter is constituted of polyethylene foil.
The formulas for calculating the temperature-time curve are derived from the partial differential equation for the instantaneous heat conduction:
(I) in which T is the local temperature, x is the position coordinate in the direction of the maximum temperature gradient, t is the time and a is the coefficient of tempera-ture conductivity.

i85~

The boundar~ conditions in the coolant are taken in~o consideration in the oll~wing fc~xmula:

~1 ~x ~[T(xo~ z) ~ To~ (II) x a x in which To is ~he cooling tempera~ure7 3~ is ~he outer wall of the sample~ ~l,is the hea~ conduc~ y of the walï and is the heat ~ransfer coeffici~n~.

~ he other boundary conditions are ob~ained from ~he forrnula III:

1~ ~ An~L~ ~III) ~ - xik ~- x 10 xki = xik representq the location at which the two media meet~

The migration o~ the phase boun~ary in the liquid me~:ium, which corresponds to a migrating heat source7 is considered in ~he following equation:

P~at ~~ ~LI~o (IV) whereby medium i merges into medium ko ~ 15 -~3S8~if~;~

FIG, S shows th~ resul~s ob~ained with a sample having a total th~ckness o 10.8 mm in which, for clarity, the distance :frorn ~he cooled wall has been plot~ed în an expanded scale (see the x value of the absclssa). The values of ~ are thus more sharply drawnO Each cur~e corresponds ~o a given time ~. The bio~og-lcal agent is human blood admixed with 14% by weight of ~ydroxye~hyl starch as a cryogenic protective agen~. In order to keep ~he temperature ~rad~n~ as low as possibleJ as is necessary, 10 for example, ~o pro~e~: the leucocyte, ~he pla~es of low conductivity are 1~ ~mn th~ck plates of low-pressure polye~hyleneO me assembly of recep~acIe holder, biorecep-tacle containing the biological specimen and low-thermal-con-ductivity pla~es is immersed in liquid ni~rogen a~ a ~empera-~ure o ~.1196~o

Claims (11)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the deep freezing of biological sub stances which comprises the steps of:
enclosing the biological substance to be deep frozen in a bioreceptacle, determining the temperature time curve for the outer wall of said bioreceptacle which corresponds to the temperature gradient necessary to freeze all of said biological substance within the receptacle with an effective cell survival rate; and subjecting the bioreceptacle to heat exchange with a fluid coolant at a temperature sufficient to deep freeze said biological substance while controlling the temperature applied to the outer surface of said bioreceptacle as a function of time to conform to said temperature-time curve whereby the biological substance within the receptacle is subjected substantially exact-ly to said temperature gradient.

2. The process defined in claim 1 wherein said biore-ceptacle is sprayed with said liquid coolant and the rate at which said liquid coolant is sprayed is controlled in dependence upon the temperature measured. at the outer wall of said biore-ceptacle.

3. The process defined in claim 1 wherein the biore-ceptacle is immersed in the liquid coolant and the temperature at the outer surface of said bioreceptacle is controlled as a function of time by interposing between said bioreceptacle and said liquid coolant a material of low thermal conductivity and of a thickness determined by said temperature-time curve.

4. The process defined in claim 1, further comprising the step of heating the outer surface of said bioreceptacle to maintain the temperature thereof in conformity with said temperature-time curve during the deep freezing.

5. An apparatus for the deep freezing of biological substances contained in a bioreceptacle, said apparatus comprising:
means for subjecting said bioreceptacle to heat exchange with a liquid coolant at a temperature sufficient to deep freeze the biological substance therein; and means for controlling the temperature at an outer surface of said bioreceptacle as a function of time to conform with a calculated temperature-time curve providing a temperature gradient at which said biological substance is deep frozen with an effective cell survival rate.

6. The apparatus defined in claim 5 wherein said means for subjecting said bioreceptacle to heat exchange with said liquid coolant comprises a cooling channel, means for supply-ing a liquid coolant to said coolant channel and a control member between said supplying means and said coolant channel, said control means including a controller operatively con-nected to said control member for regulating the supply of the liquid coolant to said cooling channel.

7. The apparatus defined in claim 6 wherein said cooling channel is provided with spray means for spraying said liquid coolant toward said receptacle.

8. The apparatus defined in claim 7, further com-prising a holder in said cooling channel for said biorecep-tacle, said holder having a temperature-sensing element in contact with an outer surface of said bioreceptacle and operatively connected to said controller.

9. The apparatus defined in claim 8 wherein said holder is formed on a sruface opposite said bioreceptacle with heating means controlled by and connected to said controller.

10. The apparatus defined in claim 6 wherein said supplying means includes a container for the liquid coolant and a container for said coolant in gaseous form and at an elevated pressure said controller including means for interconnecting said containers upon a decrease in the pressure within the container for said liquid coolant.

11. The apparatus defined in claim 5 wherein said bioreceptacle is subjected to heat exchange with said liquid coolant by immersing said bioreceptacle in a bath of said liquid receptacle, said means for controlling the temperature at the outer surface of said bioreceptacle including plates of low thermal conductivity interposed between said coolant and a metal plate in contact with said bioreceptacle and of a thick-ness selected to maintain a temperature at said outer surface substantially in conformity with said temperature-time curve, and electrical heating means on said metal plates for fine adjustment of the temperature at said outer surface of said bioreceptacle.