WO2014111518A1

WO2014111518A1 - Tissue bioreactor

Info

Publication number: WO2014111518A1
Application number: PCT/EP2014/050899
Authority: WO
Inventors: Kristina BLOM
Original assignee: Medibiome Ab
Priority date: 2013-01-17
Filing date: 2014-01-17
Publication date: 2014-07-24

Abstract

The present invention relates to a tissue bioreactor (10)for culturing tissue explants (17) ex vivo. The bioreactor (10) comprises a chamber with a cavity comprising an open end (13) with a first inner wall diameter, a closed end (14) with a second inner wall diameter, and a ledge (15) encircling the inner wall of the chamber between the closed end (14) and the open end (13); located gel support (16) is arranged between said ledge (15) and said closed end (14) of said cavity onto which a soft tissue explant (17) is arranged. The cavity comprises an inlet (18) and an outlet (19) for continuous flow of medium through the tissue bioreactor (10). The invention also relates to a method for growing and studying a soft tissue explant exvivoin said bioreactor. The bioreactor and method of the invention is advantageously used when studying the effects and physiological processes during wound healing and/or soft tissue integration optionally after the soft tissue explant has been exposed to a physical, biological or chemical insult. (Fig 1)

Description

TISSUE BIOREACTOR

TECHNICAL FIELD

The present invention relates to a tissue bioreactor for culturing soft tissue explants, and a method for using said tissue bioreactor to study wound healing, biocompatibility and metabolic activity of soft tissue explants optionally in the presence of a microbial burden, wound dressings, test compositions and/or agents.

BACKGROUND OF THE INVENTION

The predictability of a method is central in foretelling how products such as wound dressings or products exposed to soft tissue integration will function as well as their biocompatibility in clinical use. Claims made for a product are continuously evaluated during the development of a product to prove "proof of concept" and conformity.

Generally, if it is a novel product, extensive preclinical testing must precede animal and clinical studies to reduce the number of animals and humans required in directives and standards. The initiative to reduce the number of studies on animals and humans owes to ethical concerns but also difficulties in managing animal and human studies in terms of access and costs.

Existing methods to study wound healing and soft tissue integration are standard test methods or methods published in peer-reviewed journals. Standard test methods to study the safety of medical devices are described in ISO 10993 Biological evaluation of medical devices. The simplest form of standard methods used is based on studies using a monolayer of cells and more complex forms uses animal studies. In an effort to replace animal studies to evaluate safety, the EU commission has started a center, European Center for the Validation of Alternative Methods (ECVAM). This initiative along with other ongoing research has resulted in that synthetic tissue models including different kind of cells have been developed.

Recently developed systems have used cultured human epidermal cells grown on one of several possible substrates with the aim to mimic the dermis of the skin. These systems have a high level of sophistication and are available commercially, but at a relatively high price. Furthermore although very sophisticated these models still fail to reproduce the key function of skin accurately, i.e. they fail to generate a stratum corneum of equal permeability to real skin. This failure means that such models cannot be used to adequately evaluate effect of topically applied materials.

US20060057558 discloses a method which employs a piece of skin of an area greater than 1 cm cultured under conditions that maintains its viability and substantially normal structure for sufficient time for topically applied test device to exert an effect. The skin includes the majority of the epidermal layer plus an appropriate amount of supporting dermis. The surface of the skin is partitioned by a surface barrier film which creates a pattern of isolated regions to which different test devices can be topically applied and subsequently tested.

US201 10045477 discloses a human skin explant system and use of the system for testing the effects of compositions on the metabolic activity of the skin. Human skin explants of up to about 25 mm in diameter are placed in a medium leveled with the height of the explants. The skin explants are incubated at about 32-37°C and the culture medium is refreshed every day. Compositions may be applied to the skin and the effects of the compositions maybe analyzed through usual methods.

A human skin chamber model to study wound infection ex vivo is disclosed by

Steinstrasser et al. (Steinstresser, L, Sorkin, M., Niederbichler, A.D., Becerikli, M., Stupka, J., Daigeler, A., Kesting, M. R., Strieker, I., Jacobsen, F. and Schulte, M., Arch Dermatol Res (2010) 302:357-365). The human skin chamber (the BO-drum) is a stainless steel chamber that comprises two disk-shaped, steel plates, one base plate and one cover plate. The base plate has a diameter of 3 cm with a central opening of 1 cm and the cover plate has a diameter of 3 cm and a 4 mm inner central opening that is recessed 3.5 mm inferior to the remainder of the drum surface. The skin explant is triangulated into the base plate and the cover plate is secured without rotation force to apply tension to the cultured full skin to prevent dermal contraction due to the retraction forces of the elastic fibers and to physically support skin tissue at an air-liquid interface. Agents may be applied onto the surface of the skin explant which thereafter is cultured at the air-liquid interphase. The human skin chamber enables wound healing studies by providing two compartments that can be treated or analyzed separately.

Wound healing is complicated by the presence of a microbial burden which has recently been discussed to exist as a microbial biofilm. Hence, it is being realized that wound healing studies shall be done in presence of a microbial biofilm, which is done in animal studies and most often in pigs. Lipp et al. discloses a method for testing the effects of wound dressings on bacterial bioburden in vitro (Lipp et al. Testing wound dressings using an in vitro wound model, J Wound care. 2010 June; 19(6): 220-226). However, this in vitro method can't analyze both the activity of biofilm and wound healing in the same assay. Soft tissue based assays are clinically more relevant since biofilm can exist not only on the materials, but also in surrounding tissue and thus shall be studied.

Wound healing studies to predict the effect of advanced wound dressings intended for hard to heal wounds have been problematic since monolayer cells are far from reality and animals differ from humans in terms of physiological and anatomical properties. The pig is considered to be the most closely related to humans, although the wound healing processes are much quicker and facilitated by contraction that does not occur in humans. Furthermore, even though the pig is not as furry as rodents, it is still furrier than humans.

There is thus a need for a culturing device as well as a method for culturing soft tissue that has the potential to replace animals that today are used for studying wound healing and soft tissue integration. Advantageously, the inventive device can also be used to evaluate biocompatibility, i.e. if a product is tissue friendly or will provoke a toxic effect. It is therefore the object of the invention to provide a soft tissue device and a method for culturing soft tissue explants that will have the potential to complement, or even replace, clinical phase I studies proving safety and clinical phase II studies indicating function in humans.

SUMMARY OF THE INVENTION

This object is achieved by a bioreactor optimized for culturing soft tissue explants, said bioreactor comprising a chamber with a cavity comprising an open end with a first inner wall diameter, a closed end with a second inner wall diameter, and a ledge encircling an inner wall of the chamber between the open end and the closed end. A gel support is arranged between the ledge and the closed end of said cavity onto which the soft tissue explant is arranged. The cavity comprises an inlet and an outlet to supply a continuous flow of medium through the tissue bioreactor.

Advantageously said first inner wall diameter is greater than said second inner wall diameter, thereby forming the ledge which is arranged at a transition between said first inner wall diameter and said second inner wall diameter.

As used herein the term "tissue explants" generally refers to tissues that connect, support, or surround other structures and organs of the body. Preferably the tissue is a soft tissue which includes but is not limited to mucosa, tendons, ligaments, fascia, skin, fibrous tissues, fat, synovial membranes, muscles, nerves and blood vessels. Advantageously the bioreactor is used for culturing skin explants, obtained from mammals. The term "mammal" includes, but is not limited to, humans, nonhuman primates such as

chimpanzees and other apes and monkey species, farm animals such as cattle, sheep, pigs, goats and horses, domestic mammals such as dogs and cats, laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In the most preferred embodiment, the subject is a human.

A continuous flow of medium through the bioreactor will provide a more authentic, in vivo testing environment for the tissue explant than existing in vitro models. Topically applied agents such as bioactive substances, and/or topically applied devices such as wound dressings and implants can be analyzed under continuous supply of fluids such as wound fluid, blood, urine or saliva that together with the supplied medium will support the tissue and its viability for extended time, as well as expose the tested agent and/or device with in vivo relevant fluid underneath the tissue.

The outlet from the cavity has an outlet diameter, and the inlet to the cavity has an inlet diameter. To enable a continuous flow of medium through the tissue bioreactor, as well as to maintain the tissue explant in liquid air interphase, the diameter of the outlet through which waste products are transported away from the bioreactor is larger than the diameter of the inlet through which fresh medium is supplied to the bioreactor. Advantageously the outlet diameter is 1.2-1 .8 times, more preferably 1 .4 -1 .6 times, most preferably 1.5 times larger than the inlet diameter.

In order to maintain viability of the tissue explant, the tissue explant must be cultured in the air-liquid interphase. In the bioreactor of the invention this is solved by the

arrangement of a gel support in the cavity, onto which the tissue explant is arranged. Advantageously the gel support is a nutrient gel that is individually tailored to suit the needs of the tissue explant being cultured in the bioreactor. Said gel support is designed to fit into the cavity, filling said cavity leaving only a narrow space around the gel support.

The tissue explant is advantageously cut to a size such that the area of the bottom side of the tissue explant will substantially equal the area of the top surface of the gel support. Normally a tissue explant has two surface areas, an upper surface and a bottom side surface. The nature of the soft tissue explant will determine which side of the tissue explant that will be the "bottom side" or the "upper side". However, if the soft tissue explant is derived from skin, the "upper side" of the soft tissue explant will normally be the side with the outermost layers of the skin, and the "bottom side" will be the side of the skin normally facing the inside of the body.

The liquid medium will enter through the inlet and flood said cavity, filling any void around the gel support. The gel support is rigid enough to support the tissue explant, yet porous enough to let medium enter and diffuse through its pores. The height of the gel support is advantageously sized such that the top surface of the gel support coincides with the inlet and outlet of the cavity, thereby enabling the medium to surround and diffuse into and through said gel support, but more importantly the liquid medium will enter the narrow space between the gel support and the bottom side of the tissue explant. When arranged on top of the gel support, the upper surface of the tissue explant will be exposed to the air, while the bottom side of said tissue explant will be exposed to a continuous flow of medium. As a consequence the tissue explant will be grown in the air-liquid interphase during a continuous flow of medium. The continuous flow of medium will surround the bottom side of the tissue explant and remove any waste products from the tissue explants (e.g. cell debris and metabolic end products) and upon microbial presence,

microorganisms that have not adhered to tissue or biomaterial. Said waste products will exit through the outlet.

The design of the bioreactor of the invention is very advantageous for evaluating different effects of topically applied devices during e.g. wound healing. As used herein the term topically applied devices encompasses any kind of wound dressings such as bandages, plasters, coverings, gauzes, pressure bandages, as well as such devices containing bioactive agents. The continuous flow of a medium as provided in the bioreactor of the present invention enables an experimental design which will mimic e.g. a exuding wounds, an oral environment for a dental implant, a lens of an eye or a urinary catheter. Such environments are known for persistent and complicated infections.

Generally many official standard methods are based on using a topically applied device of a certain size or weight, which means that the device is cut or punched in order to provide the correct sample size. Such cutting or punching may cause problems when the device contains bioactive substances, as such a substance tends to leak from the cut edge instead of through the surface facing the tissue or wound.

The problem of leakage of bioactive substances from the cut edges on topically applied devices, such as e.g. wound dressings, is solved in the bioreactor of the present invention by arranging a test device retainer in the chamber at the open end with a first diameter. The test device retainer has a general shape and size which makes it fit snugly into the chamber with the first inner wall diameter. The test device retainer is advantageously provided with a through bore and has an upper end and bottom end. The upper end of the test device retainer is provided with a rim having a diameter that is larger than the inner wall diameter of the chamber with an open end. The bottom end of the test device retainer is advantageously provided with a chamfered lower edge. When the test device retainer is inserted into the chamber with an open end, the chamfered lower edge will press the edges of the topically applied device, e.g. a wound dressing against the ledge, sealing said edges tightly and thereby preventing any substances or agents from leaving the wound dressing from anywhere but the surface exposed to the tissue explant.

A rod may be arranged to fit into the through bore of the test device retainer, and said rod may be provided with a weight. The rod may be used to simulate the effects of pressure of biomaterials on tissue. Weights equal to about 20-60 mm Hg may be applied to the rod. Such weight may be applied e.g. when mimicking the pressure applied on venous leg ulcers.

The tissue bioreactor is advantageously placed in an incubator providing an atmosphere of preferably about 5% C0₂, and >95% humidity.

A further aspect of the invention relates to a method for culturing and/or studying tissue explant in the bioreactor of the invention. Said method comprises the steps of

- placing a gel support into the cavity;

- placing a tissue explant on top of said gel support;

- providing a continuous inlet flow of medium through the inlet, and a continuous outlet flow of waste material through the outlet of the cavity.

The flow rate of medium through the bioreactor is advantageously from 0.1 ml/hour to about 10 ml/hour, more preferably between 0.3 - 0.5 ml/h.

Said method for culturing tissue explants is advantageously used for studying different aspects of tissue explants. During the culturing process one or more samples may be obtained from any one of the gel support, the tissue explant, the waste material and/or the medium as well as applied biomaterials and/or agents, for use in such studies. The evaluation and analysis of such samples are well known to the person skilled in the art, and will therefore not be described here. The method of the invention may further include a step of subjecting the tissue explant to an insult. The insult may be a defined biological, physical, and/or chemical insult which is applied prior to, or during the culturing process. Such an insult normally results in some kind of damage to the tissue explant.

The step of subjecting the tissue explant to an insult is particularly useful when it is desired to evaluate an effect of a topically applied agent or topically applied device on the response to the biological, physical or chemical insult. Thus the method of the invention includes culturing of tissue explants that have been subjected to some kind of insult, and optionally thereafter exposed to a topically applied agent or topically applied device. As used herein the term topically applied agent is intended to encompass any topically applied compositions such as ointments, creams, lotions, emollients, liniments and gels with or without bioactive ingredients. Topically applied devices are intended to encompass any kind of biomaterial such as e.g. wound dressings, bandages, plasters, coverings, gauzes, pressure bandages including such biomaterials with, or without bioactive agents. An example of a biological insult is the inoculation onto the surface of the tissue explant of microorganisms, and under certain circumstances, a source of nutrient for these microorganisms. Thereafter one or more topically applied agents and/or materials may be applied to evaluate their ability to reduce growth of the microorganisms on the tissue explant and/or their ability to modify the response of the tissue explant to the

microorganisms

Another example of a biological insult is the inclusion in the culture medium maintaining the tissue explant of a biologically active substance that induces a change in the tissue explant. Many biologically active molecules can be used including, but not limited to interleukins, growth factors, tumor necrosis factors, histamine, prostaglandins, nitric oxide, insulin, insulin like growth factors etc. This system can then be used to determine the ability of topically applied devices to reduce the response of the tissue explant.

An example of a physical insult is the exposure of the tissue explant to irradiation, such as e.g. ultraviolet light through the use of a controllable solar simulator that provides a measured dosage of radiation. Here the system is especially useful to compare the efficacy of different test agents in reducing the damage done to the tissue explant, or the response of the tissue explant to the ultraviolet light. The tissue explant can be irradiated before or after application of the surface barrier film and the topically applied agents and/or devices applied before or after the irradiation. Another example of physical insult may be an infliction of a wound to the tissue explant, such as scratching or cutting the tissue explant with a sharp object, or by burning the tissue explant with a heated object. Either the effect of the inflicted wound itself may be evaluated, or a topically applied agent and/or material as listed above may be applied to relieve the effect induced by such wound infliction. In one embodiment the inflicted wound may be inoculated with one or more microorganisms, and thereafter the effects of an application of a topically applied agent and/or device to said wound, or the absence thereof, is evaluated.

Another example of physical insult may be tissue integration, wherein e.g. an implant is integrated into the tissue explant. Either the effect of the integration itself may be evaluated, or a topically applied agent and/or material as listed above may be applied to relieve the effect induced by such tissue integration. In one embodiment the tissue integration of an implant may be accompanied by an inoculation with one or more microorganisms, and thereafter the effects of an application of a topically applied agent and/or material to the integration site, or the absence of such an agent/device, is evaluated.

An example of a chemical insult is the exposure of part of or the whole tissue explant surface to materials having irritant and/or sensitizing properties. Examples of materials having irritant properties include but are not limited to solvents, surfactants, acids, toxins, biocides, agents, textiles, chemicals, cosmetics, hygiene products, and biomaterials. Examples of materials having sensitizing properties are compounds capable to induce an adaptive immune response through skin contact, e.g. allergens such as food allergens such as for example milk, soy, eggs, wheat, peanut, tree nuts, fish and shellfish, non-food allergens, such as e.g. latex, toxins interacting with proteins such as toxins from poisonous plants and/or certain chemicals. Either the effect of the irritant and/or sensitizer itself may be evaluated, or after a topically applied agent and/or device has been applied to relieve the effect induced by such treatments with irritants and/or sensitizers. In one embodiment the exposure to an irritant or sensitizing material may be accompanied by an inoculation with one or more micro-organisms, and thereafter the effects of an application of a topically applied agent and/or device to the exposed site, or the absence of such an agent/device, is evaluated.

When topically applied devices are applied to the tissue explant, the test device retainer having a bottom end with a chamfered edge is advantageously fitted into the chamber with an open end and a first inner wall diameter, said chamfered edge wedging the topically applied device to the ledge. By wedging the edges of the topically applied device to the ledge encircling the inner wall of the chamber at the transition between the first inner wall diameter to the second inner wall diameter, any agents incorporated into the topically applied device is prevented from escaping through said edges and will diffuse from the surface in contact with the tissue explant instead.

When pressure bandages are tested, a rod optionally provided with weights may be fitted into the through bore of the test device retainer thereby providing physical pressure onto the topically applied device. Pressure may also be applied to implants integrated into tissue.

A further aspect of the present invention relates to a use of the bioreactor of the invention for culturing and/or studying of tissue explants ex vivo. The bioreactor is particularly useful when studying the behaviour of tissue explants after exposing said tissue explant to some kind of insult, such as a defined biological, physical, or chemical insult which is applied prior to, or during the ex vivo culturing process. Examples of such defined biological, physical, or chemical insults are listed above.

The bioreactor may be used in the evaluation of a healing process after infliction of such insults to the tissue explant. However, the bioreactor of the invention is particularly advantageous during the evaluation of an effect of a topically applied agent or topically applied device on the response to the biological, physical, or chemical insult.

Advantageously the bioreactor of the invention is fitted with a microscope to enable in situ analysis in ex vivo for screening soft tissue exposure. For example a Multi-photon fluorescence microscope (MPM) is particularly useful for non-invasive imaging. MPM allows for 3D-visualisation of biological tissue at the cellular level in a non-destructive fashion and cellular responses may be monitored continuously. For example, soft tissue explants such as skin may be exposed to sensitizers or irritants using the bioreactor of the invention and the response in skin in situ can be studied on-line using MPM. The tissue may subsequently be sectioned and analyzed histologically and immunohistologically to study toxic effects. Furthermore, the inflammatory response can be analyzed on protein and gene expression levels to evaluate the physiologic response such as inflammatory factors and growth factors.

Wounds are an ideal environment for bacterial colonization and biofilm formation. The wound bed provides both a surface on which to grow, as well as being an ample supply of nutrients. A biofilm when used in the context of the present invention is generally intended to mean an aggregate of microorganisms in which cells adhere to each other. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living tissue or inanimate surfaces. The microbial cells growing in a biofilm are

physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium.

The bioreactor of the invention is advantageously used when studying the effects on biofilm and surrounding tissue during wound healing and/or soft tissue integration. For example, soft tissue explants subjected to some kind of insult as listed above, and subsequently exposed to inoculation of one or more microorganisms forming a biofilm therein. The tissue explant may also be exposed to some naturally occurring body fluid, such as wound fluid, urine, blood, saliva, feces, and/or seamen. The tissue explant subjected to such treatments may thereafter be evaluated before and/or after application of a topically applied agent or device. One such example may be dental implant abutments wherein a part of the abutment will be integrated in the tissue and a part will be above the tissue exposed to air. The abutment and tissue can be flooded by saliva with or without bacterial biofilm.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a perspective view of the bioreactor of the invention.

Figure 2 is a schematic view of the different parts of the bioreactor of the invention including a tissue explant and a topically applied device before assembly.

Figure 3 is a perspective view of the bioreactor when assembled.

Figure 4 is a top view of the bioreactor of the invention.

Figure 5 is a schematic view of the bioreactor when assembled as viewed from V-V in figure 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the following the invention will be described in a non-limiting way and in more detail with reference to exemplary embodiments illustrated in the enclosed drawings, in which Figures 1 -5 illustrate a tissue bioreactor 10 of the invention. Said tissue bioreactor 10 comprises a chamber 1 1 with a cavity 12. The chamber 1 1 has an open end 13 with a first inner wall diameter and a closed end 14 with a second inner diameter. The first inner wall diameter of the chamber 1 1 is greater than the second inner wall diameter of the cavity 12. A ledge 15 encircles the inner wall of the chamber 1 1 between the open end 13 and the closed end 14, said ledge 15 forming a transition from the inner wall diameter to the second inner diameter.

A gel support 16 is arranged at the closed end 14 of the cavity 12, and on top of said gel support 16 a tissue explant 17 is placed. The thickness of the gel support 16 is adjusted so as to maintain the tissue explant 17 at the air-liquid interphase (see Figure 5).

The cavity 12 comprises an inlet 18 and an outlet 19 for continuous flow of medium through the soft tissue culturing device. The inlet 18 and outlet 19 are preferably located in the upper part of the cavity 12 to ensure adequate and continuous flow of medium between the gel support 16 and the bottom side of the soft tissue explant 17.

Advantageously said outlet diameter is larger than said inlet diameter to allow a satisfactory flow through the bioreactor 10. The outlet diameter is 1.2-1.8 times, more preferably 1.4 -1 .6 times, most preferably 1.5 times larger than the inlet diameter.

The inlet 18 is connected to storage bottles for growth medium (not shown), which supplies the bioreactor 10 with e.g. growth medium suitable for growing the soft tissue explant 17. However, any kind of body fluid such as e.g. wound fluid, urine, blood, saliva, feces, and/or seamen may also be supplied through the inlet. The outlet 19 is connected to waist bottles for collecting any waste or debris formed by the tissue explant 17 or biofilm during growth. Optionally the outlet 19 may be connected to sample bottles for collecting samples to be tested. The flow of medium through the bioreactor 10 may be adjusted from 0 ml/h (i.e. substantially no flow at all) up to a maximum flow of about 10 ml/h. The flow rate through the reactor may depend on the type of explant 17 or experiment performed.

A test device retainer 20 may be fitted into the chamber 1 1 . Said test device retainer 20 has an outer wall diameter which is slightly smaller than the chamber first inner wall diameter, which will make the test device retainer 20 fit snugly into the chamber 1 1 . The test device retainer 20 has a through bore 21 with an inner wall diameter that is slightly greater than the second inner diameter of the cavity 12, which will prevent said test device retainer 20 from reaching past the ledge 15 and down into the closed end 14 of the cavity 12. Said test device retainer 20 has a top end fitted with a rim 22, and a bottom end provided with a chamfered lower edge 23. The chamfered lower edge 23 will, when the test device retainer 20 is fitted into the chamber 1 1 , wedge any topically applied device 24 such as e.g. a wound dressing (as depicted in figures 2 and 5) to the ledge 15 which encircles said inner wall of the chamber 1 1 . The cut edges on the topically applied device 24 will thereby be efficiently sealed, and any agent contained in said topically applied device 24 will be prevented from leaking through the edges and exit the topically applied device 24 from the surface instead. The outer surface of the test device retainer 20 is advantageously provided with screw threads in order for the test device retainer 20 to be secured inside the open end 13 of the cavity 12 (see figures 1 , 2 and 5. However, said test device retainer 20 may in another embodiment be secured by means of an O-ring and friction (not shown).

A rod 25 may be fitted into the through bore 21 of the test device retainer. Said rod 25 may optionally be provided with a weight of e.g. 20-60 mm Hg to simulate a pressure of the topically applied device 24 onto the tissue.

Example 1

The following example will illustrate how the bioreactor 10 of the invention may be used to establish a human ex vivo wound infection to evaluate the activity of antimicrobial wound dressings and their placebos on the formation of biofilm and the effect on surrounding tissue in regard to wound healing processes and immunological response.

Wound dressing preparation

Each wound dressing to be tested was punched into circular pieces with a diameter of 14 mm under aseptic conditions and stored in sterile petri dishes until use.

Tissue preparation

Skin explants were donated from patients undergoing breast or abdominal reduction surgery (Sahlgrenska University hospital, Goteborg, Sweden) and transported in physiological saline (0.85% NaCI). Before use, the explant was cleaned in 70% ethanol for 30 seconds and washed 3 times in 0.85% NaCI.

Organ culture

The subcutaneous fat was excised using surgical scissors under sterile conditions. The skin explant was thereafter washed thrice in 0.85% NaCI and cut into seven circular pieces using a 12mm punch. In the center of each skin explant a 1 mm deep and 3mm wide wound was created. The skin explants were transferred to a 12 well plate and 0.5-1 ml of culture medium (Dulbeco's modified eagles medium (DMEM) containing 10% Fetal bovine serum (FBS) and antibiotics (penicillin 50 μ/ml, streptomycin 50 U/ml)), depending on the thickness of the skin explant, was added to each well to culture the skin explant in the air-liquid interphase. The skin explant was incubated for 3 days at 37°C in a C0₂- Incubator (NuAire, Plymouth, MN) at 5% C0₂, >95% humidity. On day three, the culture media was changed to DMEM containing 10% FBS and 100X glutaMAX and cultured for another 3 days. For runs 1 -7 the skin explants were cultured for 10 days. Media was changed every two days.

Assessment of baseline

On day 6, one of the skin explant samples was cut into two halves. One of the two halves was cut into smaller pieces and each piece was weighed to make sure that the tissue did not exceed 30 mg. The tissue was placed in RNAIater and stored at -80°C for further gene expression analysis. The other half was nailed to a cork mat and fixed in 5 ml 4% buffered neutral formaldehyde solution and sent to HistoCenter (Goteborg, Sweden) for hematoxylin & eosin (H&E) staining.

DMEM agar preparation

1.5 g agar was added to 90 ml DMEM and dissolved at 90°C. The solution was sterilized at 120°C for 15 min and then placed in a water bath at 45°C for at least one hour. 10 ml of FBS (giving a final concentration of 10%) was added and the solution was poured in to petri dishes, 30 ml in each. Circular pieces with a diameter of 14 mm were punched under sterile conditions providing gel supports to be arranged into the cavity.

Bacteria preparation

Streak plates of Pseudomonas aeruginosa ((PA01 ), CCUG: 56489 (ATCC: 15692)) and Methicillin resistant Staphyloccus aureus ((MRSA), CCUG: 35600 (ATCC: 12600)) were made on Tryptic soy agar (TSA) (oxoid) plates and grown for 18-24 hours. One colony of each bacterium was inoculated in 5 ml of Tryptic soy broth (TSB) and grown under shaking conditions at 225 rpm and 35 ± 2°C for 18 hours. The OD was measured at 600 nm using a spectrophotometer (Genesys, Thermo Scientific) and cell concentration adjusted to 1 -5x10⁶ cells/ml using DMEM. The two bacterial species was then mixed 1 :1. Reactor preparation and wound infection

The 14 mm punched DMEM agar plug (i.e. the gel support) was placed in the cavity of the bioreactor. The skin explant was placed on top of the agar, skin side facing upward, and 300 μΙ of medium was added. The wound was infected with 10 μΙ of the mixed bacterial culture and the bacteria were allowed to equilibrate and form micro-colonies for 2 hours before the dressings were placed on top of the skin. The reactor was assembled and placed in the incubator. DMEM with 10% FBS and GlutaMAX (1000 mg/l) was pumped through the bioreactor system at 0.6 ml/h and the rectors were incubated at 37°C (5% C0₂, >95% humidity) for 72 hours.

Assessment of wound infection and wound healing

After 3 days the skin explants and dressing were removed from the bioreactors. The dressings were placed in 10 ml of Dey-Engley broth, sonicated and vortexed for 1 minute respectively. The samples were then serial diluted and spread on TSA plates to analyze the bacterial contamination level within the dressings. The skin explants were cut in halves, and one half was nailed to a cork mat and fixed in 5 ml 4% buffered neutral formalin solution for further histological analysis on wound healing processes. General hematoxylin eosin and Ki67 staining to study cell proliferation were done. The other half was cut into smaller pieces and 30 μg was placed in RNAIater and stored at -80°C for gene expression analysis of cytokines and growth factors. The remainder of the skin explant sample was homogenized in 6 ml 0.85% NaCI and serially diluted and spread on TSA plates for analysis of the microbial biofilm burden in the soft tissue.

Example 2

The following example discloses the wound healing in a skin explant infected by bacteria and cultured in the bioreactor (Fig. 6A) of the invention essentially as described in Example 1 when compared to the healing process in a skin explant infected by bacteria and cultured in a batch culture (Fig. 6B). In a batch culture there is no continuous flow of medium that will supply the soft tissue with fresh medium and remove any debris discharged from the tissue during growth and healing. As can be seen in Figure 6A the reepithelization process has started, and the amount of bacteria has decreased considerably compared to the batch cultured skin explant in Figure 6B.ln the batch cultured skin explant no reepithelization is observed and the bacterial count is still high.

Claims

A tissue bioreactor (10) for culturing soft tissue explants (17), said bioreactor (10) comprising

- a chamber with a cavity having an open end (13) with a first inner wall diameter, a closed end (14) with a second inner wall diameter, and a ledge (15) encircling an inner wall of the chamber between the closed end (14) and the open end (13); and

a gel support (16) arranged between said ledge (15) and said closed end (14), said gel support having a top surface onto which a soft tissue explant is arranged,

characterized in that

said cavity (12) comprises an inlet (18) and an outlet (19) for continuous flow of medium through the tissue bioreactor (10).

The bioreactor (10) according to claim 1 , wherein said first inner wall diameter is greater than said second inner wall diameter.

The bioreactor (10) according to claim 2, wherein said ledge (15) is arranged at a transition between said first inner wall diameter and said second inner wall diameter.

The bioreactor (10) according to any one of claims 1 -3, wherein the tissue explant (17) is chosen from the group consisting of mucosa, tendons, ligaments, fascia, skin, fibrous tissues, fat, synovial membranes, and/or muscles.

The bioreactor (10) according to claim 4, wherein the tissue explant (17) is a skin explant and/or a mucosa explant.

The bioreactor (10) according to any one of claims 1 -5, wherein the outlet (19) from the cavity has an outlet diameter and the inlet (18) to said cavity has an inlet diameter, said outlet diameter being larger than the inlet diameter.

The bioreactor (10) according to claim 6, wherein the outlet diameter is 1.2-1 .8 times, more preferably 1 .4 -1 .6 times, most preferably 1 .5 times larger than the inlet diameter.

8. The bioreactor (10) according to anyone of claims 1 -7, wherein the upper surface of the gel support (16) coincides with the inlet (18) and the outlet (19) of the cavity.

9. The bioreactor (10) according to anyone of claims 1 -8, wherein the gel support (16) is a nutrient agar gel.

10. The bioreactor (10) according to anyone of claims 1 -9, wherein a test device retainer (20) having a through bore (21 ) is arranged to fit into the chamber at the open end (13) with a first diameter.

1 1 . The bioreactor (10) according to claim 10, wherein said test device retainer (20) has an upper end and a bottom end, said bottom end is provided with a chamfered lower edge (23).

12. The bioreactor (10) according to claims 10 or 1 1 , wherein a rod (25) is

arranged to fit into the through bore (21 ) of the test device retainer (20).

13. The bioreactor (10) according to claim 12, wherein the rod (25) is provided with a weight.

14. The bioreactor (10) according to claim 13, wherein the weight is equal to 20- 60 mm Hg on the soft tissue.

15. A method for culturing and/or studying tissue explants (17) in the bioreactor (10) of claims 1 -14,

wherein said method comprises the steps of

- placing a gel support (16) into the cavity;

- placing a tissue explant (17) on top of said gel support (16);

- providing a continuous inlet flow of medium through the inlet (18), and a continuous outlet flow of waste material through the outlet (19) of the cavity.

16. The method according to claim 15, wherein the inlet flow of medium is from

0.1 ml/hour to about 10 ml/hour.

17. The method according to anyone of claims 15-16, wherein the method further comprises obtaining one or more samples from the gel support (16), the tissue explant (17), the waste material and/or the medium.

18. The method according to anyone of claims 15-17, wherein the method further comprises subjecting the tissue explant (17) to an insult.

19. The method according to claim 18, wherein the insult is a defined biological, physical, or chemical insult.

20. The method according to claim 19, wherein a biological insult is the exposure of the tissue explant (17) to an inoculation with one or more micro-organisms, and/or exposure to one or more bioactive molecules.

21 . The method according to claim 19, wherein a physical insult is chosen from the group consisting of exposure of the tissue explant (17) to ultraviolet light, wounds inflicted by scratching the tissue explant (17) with a sharp object, wounds inflicted by burning the tissue explant (17) with a heated object, and/or soft tissue integration.

22. The method according to claim 21 , wherein the wound is inflicted by

scratching the tissue explant (17) with a sharp object.

23. The method according to claim 22, wherein the wound is inflicted by burning the tissue explant (17) with a heated object.

24. The method according to claim 19, wherein a chemical insult is the exposure of the tissue explant (17) to a material with irritant properties chosen from the group consisting of solvents, surfactants, acids, biocides, agents, textiles, chemicals, cosmetics, hygiene products, and biomaterials and/or toxins.

25. The method according to claim 19, wherein a chemical insult is the exposure of the tissue explant (17) to a material with sensitizing properties chosen from the group consisting of food allergens, non-food allergens, toxins interacting with proteins and/or chemicals.

26. The method according to anyone of claims 15-25, wherein the tissue explant (17) is exposed to one or more topically applied agents and/or one or more topically applied devices (24).

27. The method according to claim 26, wherein said topically applied agent is chosen from the group consisting of ointments, creams, lotions, emollients, liniments and/or gels, with or without bioactive ingredients.

28. The method according to claim 26, wherein said topically applied device (24) is chosen from the group consisting of wound dressings, bandages, plasters, coverings, gauzes, and/or pressure bandages, with or without bioactive agents.

29. The method according to any one of claims 15-28, wherein a test device retainer (20) having a bottom end with a chamfered edge (23) is fitted into the chamber with an open end (13) and a first inner wall diameter, said chamfered edge (23) wedging said topically applied device (24) to the ledge (15).

30. The method according to claim 29, wherein a rod (25) provided with a weight is fitted into the through bore (21 ) of the test device retainer (20) thereby providing physical pressure onto said topically applied device (24).

31 . Use of the bioreactor (10) according to claims 1 -14 for ex vivo culturing and/or studying of tissue explants (17) according to the method of anyone of claims 15-29.