CZ36430U1 - Device and kit for preparing a therapeutic preparation based on a carrier seeded with cells - Google Patents

Description

Device and kit for the preparation of a therapeutic preparation based on a carrier seeded with cells

Field of technology

The technical solution relates to a device and kit for the preparation of a therapeutic preparation based on a carrier seeded with cells.

Current state of the art

In human medicine, the safety and efficacy of mesenchymal stromal cells (MSCs) for the treatment of various diseases and injuries are increasingly being verified in clinical trials. Clinical studies using cell therapies and tissue engineering focus, among other things, on the treatment of neurodegenerative diseases, autoimmune diseases, cardiovascular diseases, diabetes, diabetic foot syndrome, orthopedic applications and others. MSCs can also be used for allogeneic administration, i.e. using cells from a healthy donor to treat patients who cannot use their own MSCs.

Before application, the product intended for applications in human medicine must undergo safety tests that last 48 to 72 hours. Throughout safety testing, the product must be kept in a closed sterile environment and must not be handled until use in the operating room.

Cryopreservation represents the only possibility to ensure the rapid availability of cell graft for applications in tissue engineering as well as to ensure the safety and quantity of the product during repeated administrations. In this case, the cryobank contains a set of tubes with the same cells from one batch, some of which are intended for quality testing that can be performed at different time intervals during the storage period. Under optimal validation conditions, the results of these quality tests can be transferred to final batches of product used for patient application without further evaluation of the final product.

However, for the cryopreservation of different types of cells, it is necessary to use cryoprotectants, which prevent the formation of ice crystals during freezing, thereby protecting cells from membrane damage and cell death. Currently, dimethyl sulfoxide (DMSO) is used as a cryoprotectant in most cases in relatively high concentrations (up to 10% by volume). At these concentrations, DMSO at plus temperatures has a toxic effect on thawed cells as well as on the cells and tissues of the patient to whom the cells in solution with DMSO are applied. To ensure therapeutic results and minimize the side effects associated with the application of a solution containing a cryoprotectant, it is highly desirable to develop procedures to reduce the concentration of DMSO in the freezing solutions or controllable procedures to remove DMSO from the medium after thawing the cells. In addition, the persistent presence of DMSO during in vitro culture limits the lifespan of cells and their ability to attach to biomaterial carriers. Therefore, DMSO should be completely removed from the cells before application if possible.

According to current regulations, one of the ways to remove DMSO, i.e. placing cells in or on a biomaterial carrier, should take place in a certified laboratory. This highly time- and financially costly step significantly limits the use of cryopreserved cells for their use in cell therapies and regenerative medicine. DMSO can also be removed by centrifugation of thawed cells directly in the operating room, but this increases the risk of contamination of these cells and increases the demands on operating room equipment.

A more promising way of using cryopreserved cells is to ensure the removal of DMSO in closed systems that allow seeding on a carrier, culture and subsequent handling of the cells and prevent contamination of the cell product or loss of cell function.

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Current methods for removing DMSO (and other cryoprotectants) in closed systems are developed only for blood products, where the volume of the cryopreserved blood sample is significantly higher than the volume of the preserved cells, and these systems are not applicable to small volume samples. Moreover, in addition to the already mentioned limitations regarding the size of samples, the high cost of acquiring and operating these devices and the impossibility of using them for seeding and culturing cells on/in carriers minimizes their use for the above-mentioned purposes.

Therefore, systems are being sought that would be able to safely remove DMSO (or other cryoprotectant) without large manipulations with thawed cells and ensure the planting, anchoring, cultivation, storage and transport of cells in or on biomaterial carriers and maintain the safety of the cell product.

Even in the case of freshly collected cells that have not gone through the freezing phase, the product intended for applications in human medicine must undergo safety tests that last 48 to 72 hours before use. Throughout safety testing, the product must be kept in a closed sterile environment and must not be handled until use in the operating room.

The presented technical solution aims to eliminate the problems of existing solutions.

The essence of the technical solution

The presented technical solution provides a chamber and a kit for the preparation of a therapeutic preparation based on a carrier seeded with cells, which preserve the safety of the cell product, prevent contamination from the external environment, and allow to ensure under these aseptic conditions thawing, washing of cryoprotectants, anchoring, cultivation, storage and transport of cells in or on biomaterial carriers for clinical applications. Both fresh and cryopreserved cells can be used.

In the first aspect, the technical solution provides a chamber for the preparation of a therapeutic preparation, wherein this chamber contains at least one container. The container is equipped with at least two inlet/outlet ports adapted for aseptic introduction and withdrawal of liquids, with at least one of the ports equipped with a three-way tap, one inlet of which is equipped with a filter for cleaning the incoming gases and the other inlet is closed with another valve, and while the other inlet/outlet ports they can optionally also be fitted with valves. The container is equipped with at least one closable opening for introducing and removing the carrier.

The container is gas-tight and liquid-tight. A carrier is inserted into the container and the medium and cells are introduced, the cultivation of cells also takes place in it.

The container of the chamber is made of a material suitable for contact with the cells, preferably of a material selected from polystyrene, glass, polyethylene. It can be produced using many techniques, for example 3D printing or pressing. The chamber may be reusable, in which case the material should be sterilizable.

Inlet/outlet ports are adapted for aseptic introduction and withdrawal of fluids. There are preferably two to six input/output ports, more preferably two to four. The input/output ports can be fitted with Luer-Lock adapters. The inlet/outlet ports can also be equipped with filters and/or valves and/or a three-way tap, these components can preferably be connected via Luer-Lock adapters.

The closable opening for introducing and removing the carrier may in some embodiments be provided with a screw cap, which may further be provided with a three-way tap with a filter and/or valve (this three-way tap may or may not be the only three-way tap of the vessel). In other embodiments, the closable opening for introducing and removing the carrier can be provided with a sealing lid, which can be attached to the container with, for example, clamps.

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The filter is preferably a filter with a mesh size of no more than 0.22 μτη.

In some embodiments, the chamber may further be provided with an inlet and outlet connected to a peristaltic or injection pump. This arrangement allows a dynamic exchange of the medium inside the chamber.

In the second aspect, the subject of the technical solution is a set for the preparation of a therapeutic preparation, containing the chamber described above, and also a container with a 1 to 10% (wt.) aqueous solution of CaCL.

Further, the kit may optionally include at least one additional component selected from:

- containers with 0.9% (wt.) NaCl aqueous solution or Ringer's solution;

- a carrier, preferably located in the container of the chamber;

- containers with platelet lysate or blood plasma;

- containers with dimethyl sulfoxide (DMSO).

Furthermore, for the sake of completeness, the procedure for preparing a therapeutic preparation based on a carrier seeded with cells using a chamber and a kit according to the presented technical solution is described here. A carrier intended for seeding with cells is introduced into the container of the chamber and the chamber is closed. Cells re-suspended in platelet lysate or blood plasma along with 1 to 10% (wt.) CaCl aqueous solution are then introduced into the chamber vessel through the inlet/outlet port and the chamber is left for 5 to 10 minutes at 30 to 40°C for occurrence of fibrinogen gelling reaction. The created gel attaches to the cells on the surface of the carrier or in the carrier. This creates a therapeutic preparation consisting of a carrier, cells and a gel (fibrin hydrogel).

Human platelet lysate or human plasma contain fibrinogen, which forms a gel after the introduction of CaCL solution. This gel (fibrin hydrogel) helps the retention/adhesion of cells on/in the carrier.

The carrier can preferably be washed with sterile physiological or Ringer's solution or complete culture medium before introducing the cells.

Cells can be resuspended fresh (unfrozen) with platelet lysate or blood plasma, or freshly thawed cells can be used where the platelet lysate or blood plasma was part of the cryoprotectant together with the cryoprotectant. The cryoprotectant can preferably be 1 to 10% (vol.) dimethyl sulfoxide, which minimizes cell damage during freezing, but is cytotoxic for cells in the unfrozen state, depending on the dose.

In the case of using thawed cells and introducing them into the chamber container together with the cryoprotectant, a 0.9% (mass.) NaCl aqueous solution or Ringer's solution is then introduced into the chamber container via the input/output port in order to wash away the toxic dimethyl sulfoxide for the cells, which is subsequently the input/output port will remove. This is preferably done by warming a container of 0.9% (wt) NaCl or Ringer's solution to 30-40°C and carefully injecting the solution into the chamber container via the inlet/outlet port to dilute it DMSO. Using a sterile syringe, fluid is removed from the chamber, again through the inlet/outlet port. The step of introducing 0.9% (wt.) NaCl aqueous solution or Ringer's solution and removing it can be repeated several times, usually it is repeated 1 to 4 times.

In the cell thawing and gel formation step, the cells are thawed at 30 to 43 °C for no more than 5 minutes, 1 to 10% (wt.) CaCL solution is drawn into a syringe and the thawed cells

-3 CZ 36430 UI in cryoprotective medium are collected in the same syringe. Immediately thereafter, the contents of the syringe are slowly injected into the chamber container through one inlet/outlet port. The chamber is placed for 5 to 10 minutes in a thermostat at a temperature of 30 to 40°C to allow the fibrinogen gel reaction to take place.

After the gelation reaction has taken place and the DMSO has been washed off in the case of the addition of thawed cells, or after the gelation reaction has taken place in the case of the addition of fresh cells resuspended in platelet lysate or blood plasma, the carrier with attached cells and gel (therapeutic preparation) is either ready for application or intended for further cultivation of cells on a carrier and subsequent application. Further cultivation of the cells on the carrier can last from 6 hours to 14 days, preferably 1 to 7 days. Cultivation usually takes place in a complete culture medium suitable for the cultured cell type, at a temperature of 35.5 to 39.5 °C, in a humidified atmosphere with controlled gas exchange with 3 to 7% (vol.) CO2 and 1 to 21% (vol. ) O2 in a gas mixture.

In a hermetically sealed chamber in the presence of culture medium, the cells fixed in the gel on the carrier can be kept at a temperature of 10 to 30 °C, preferably 23 to 28 °C, without access to air for up to 10 days, preferably 2 to 6 days, without any further manipulation in an aseptic environment.

The carrier is usually formed from a biomaterial and is preferably porous. Biomaterials in quality for clinical applications are particularly suitable. These are in particular synthetic biocompatible polymers such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), polyethylene glycol (PEG), polycaprolactone (PCL), polyhydroxybutyrate (PHB); also natural polymers, proteins and polysaccharides such as collagen, type I collagen, gelatin, alginate, agar, hyaluronic acid, sodium hyaluronate, hyaluronic acid benzyl ester, fibrin, chitosan, cellulose, starch; or extracellular matrix (ECM), silicone, hydroxyapatite, and mixtures of these materials. The carrier material can be in the form of hydrogel, nanofibers, microfibers, membranes or contact lenses. The carrier can be both biodegradable and non-degradable material.

The cells are adherent cells. In particular, cells are used from the group comprising mesenchymal stromal cells, for example obtained from bone marrow, adipose tissue, peripheral blood, hair follicles, epidermis, testes, umbilical cord blood, umbilical cord tissue, placenta, amniotic fluid, tissue-specific cells, for example nerve cells, limbal , endothelial, chondrocytes, fibroblasts, osteoblasts, as well as embryonic stem cells, extraembryonic stem cells and induced pluripotent cells.

The dimensions of the chamber and its container can be chosen according to the size and number of carriers that will be inserted into them. The chamber and cultivation container can be, for example, circular, rectangular or square.

The chamber and kit according to the presented technical solution can be used for washing DMSO from thawed cells attached to the carrier or in the carrier, for culturing cells on the carrier or in the carrier, for long-term preservation of cells in the carrier or in the carrier (up to 10 days) in a closed aseptic environment without air access at a temperature of 10 to 30 °C. A carrier seeded with cells in a fibrin hydrogel constitutes a therapeutic preparation.

Clarification of drawings

Giant. 1 shows the first embodiment of the chamber according to the technical solution.

Giant. 2 and 3 show the second embodiment of the chamber according to the technical solution.

Giant. 4 shows the dependence of the viability/proliferation rate of hWJ-MSC cells in hyalofast (HF, dashed line) and chondrotissue (CHT, solid line) carriers on culture time.

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Giant. 5 shows the viability/proliferation rate of hWJ-MSC cells in hyalofast (HF) carriers in both tested chambers, when stored for 0, 2, 6 days, the chambers were hermetically sealed at a temperature of 25 °C (art. Č2 - chamber no. 1, chamber No. 2; dO - cells anchored in the HF carrier after eight days of cultivation in a controlled-atmosphere thermostat before storage in a closed chamber without the access of gases at a temperature of 25 °C; d2 - cells anchored in the HF carrier after two days of storage in a closed chamber without the access of gases at at a temperature of 25 °C and d6 - cells anchored in the appropriate carrier after six days of storage in a closed chamber without the access of gases at a temperature of 25 °C).

Examples of implementing a technical solution

Example 1: Chamber construction

The chamber described in this example is shown in FIG. 1. The chamber contains chamber container A, which is provided with two inlet/outlet ports P1 and P2 for aseptic introduction and withdrawal of fluids. The container A of the chamber is further provided with an opening O, which can be closed by a screw cap U, the screw cap U is equipped with a port P3 with a three-way closable tap K, one inlet of which is equipped with a closable valve V3 and the other inlet of which is equipped with a filter F for cleaning the incoming gases , while the input/output ports P1, P2 can optionally also be provided with valves VI, V2, or can be equipped with filters (not shown).

A biomaterial carrier N is introduced into the container A of the chamber with an opening O. After the carrier is introduced, the chamber is closed with a screw cap U with a three-way closable cock K. After opening the valve VI with the input/output port Pl, Ringer's solution is injected with a syringe and the valve VI is closed. After 10 minutes, Ringer's solution is aspirated with a syringe through valve V2 through inlet/outlet port P2 and the valve is closed. Thus, the system is ready for introduction of cells together with platelet lysate or blood plasma and CaCL solution, which is introduced into the system with a syringe after opening valve V3 through inlet/outlet port P3 of three-way stopcock K.

Example 2: Chamber construction

The chamber described in this example is shown in FIG. 2 and 3. The chamber contains the container A of the chamber, which is provided with four inlet/outlet ports P1, P2, P3, P4 adapted for aseptic introduction and withdrawal of fluids. The container A of the chamber is further provided with an opening O covered by a lid B and a silicone gasket C, while the lid B is fixed by four clamps D. The input/output ports Pl, P2, P3, P4 are provided with three closable taps KI, K2, K3, K4 , one inlet of which is always equipped with a filter Fl, F2, F3, F4 for cleaning the incoming gases and the other inlet of which is equipped with a valve VI. V2. V3. V4.

Carrier N is introduced into the system through opening O after opening lid B. Carrier N completely fills the area of container A of the chamber. Alternatively, several smaller carriers (not shown) can be inserted. After introducing the carrier N, a silicone seal C and lid B are placed on the container A of the chamber, which is fixed using four clamps D. After opening any valve VI, V2, V3 or V4, Ringer's solution and valve VI are introduced into the container A of the chamber. V2. V3 or V4 closes. After 10 minutes, valve VI. V2, V3, or V4 opens and Ringer's solution is aspirated from the chamber. This is how the system is prepared for the introduction of cells together with platelet lysate or blood plasma and CaCL solution.

Example 3: Cryopreservation of cells used for seeding in a chamber vessel

Example 3A: Procedure for freezing Wharton's gel-derived mesenchymal stromal cells (human WJ-MSCs, hWJ-MSCs)

One million hWJ-MSCs were frozen at passage 3 in a freezing tube in one milliliter of cryopreservation medium composed of human platelet lysate (platelet lysate; DL) with 1,

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2, 5 and 10% (v/v) DMSO by slow freezing to -80 °C at a rate of approx. 1 °C per minute in a CoolCell freezing container, and then stored in a liquid nitrogen container. Frozen samples were removed from nitrogen after 30 days and immediately thawed at 37°C for cell viability analysis.

At 2% (v/v) and higher concentration of DMSO in the cryopreservation medium, cell membrane integrity (one of the signs of cell viability) after thawing was very high (90 to 98%) after staining with trypan blue. Even at a 1% (vol.) concentration of DMSO, the viability was found to be sufficient (65 ± 18%) (Tab. 1). However, data obtained immediately after thawing and using only one parameter (membrane integrity) may overestimate the results, as apoptotic processes that may occur during cryopreservation are known to persist approximately 24 hours after the cells are returned to physiological conditions (i.e., cell culture ). Indeed, determining the survival rate of hWJMSCs 24 h after cryopreservation as measured by the resazurin assay shows completely different results (Tab. 1), where cell viability is significantly lower compared to cell viability that was assessed by trypan blue staining immediately after thawing. The survival rate of hWJ-MSCs after cryopreservation using 1% (vol) concentration of DMSO in DL was only 6 ± 1%. Addition of 2% (v/v) DMSO to the cryoprotective medium provided 66 ± 3% cell regeneration, and application of 5% and 10% gave similar results, i.e. approximately 80% regeneration rate.

Table 1 - Cell viability and survival rate (regeneration) after thawing as measured by trypan blue and resazurin assay as a function of DMSO concentration in freezing medium prepared from human platelet lysate.

Concentration of DMSO in the cryopreservation solution Viability of WJ-MSCs WJ-MSC survival rate measured with trypan blue (in % ± standard deviation) measured by resazurin assay (in % ± standard deviation) (in%) 1 65 ± 18 6 ± 1 2 93 ±3 66 ±3 5 96 ±2 85 ± 10 10 95 ±3 79 ±9

Resazurin, a non-toxic, blue, non-fluorescent, water-soluble dye, is reduced by metabolically active cells to the pink, highly fluorescent dye resofurin. This conversion occurs only in viable cells, and therefore the amount of resorufin produced is proportional to the number of viable cells that survived cryopreservation. The resazurin assay was carried out according to the following procedure: cell constructs with biomaterial carriers were transferred for 2 hours to 0.5 ml of complete culture medium supplemented with resazurin, and then the fluorescence of the medium (Ex/Em=530/590) was measured on a Synergy TM HT Multi- Mode Microplate reader. The complete culture medium consisted of Alpha MEM medium, 5% DL and 10 pg/ml gentamicin.

Application of cryoprotective medium composed of human platelet lysate with 5% or 10% DMSO ensures high viability and survival rate of WJ-MSCs after cryopreservation.

Example 3B: Porcine fibroblast freezing procedure

Porcine fibroblasts at the 3rd passage derived from the auricle of a Czech white noble pig in the amount of 1 million cells were frozen in a freezer tube in 1 ml of a cryopreservation solution composed of porcine blood plasma with 5% DMSO by slow freezing to a temperature of -80 °C at a rate of approx. °C per minute in a CoolCell freezer, and then stored in liquid nitrogen. After thawing, the viability was found to be 91 ± 8% using trypan blue, and the survival rate measured by the resazurin test was 81 ± 10%.

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Thus, it was confirmed that the application of a cryoprotective medium composed of platelet lysate/blood plasma with 5 to 10% DMSO ensures high viability and survival rate of cells after cryopreservation.

Example 4: Washing DMSO from Cryopreserved Cells

Cells were cryopreserved as described in Example 3A. A total of 1 million cells were cryopreserved in a volume of 1 ml DL with 10% (vol) DMSO in one cryotube. Cells were thawed by rapidly warming the cryotube at 37°C. One third of the volume (330 µl, 330 thousand cells) was planted on commercial carriers of size 5x5 mm stored in chamber containers. The carriers used are commercial solid fillers used in orthopedic surgery: Chondrotissue® containing sodium hyaluronan and polyglycolic acid, and Hyalofast™ containing hyaluronic acid benzyl ester. As part of the test, 6 samples were placed from each carrier, each in a separate chamber. The supports were pre-moistened in Ringer's solution for 30 min. The Hyalofast carrier was introduced into the chamber container according to Example 1, the Chondrotissue carrier was introduced into the chamber container according to Example 2. The carriers were introduced into the chambers as described in Examples 1 and 2.

The cryotube with cells was thawed at 37°C within 5 min, 25 µl of 10% (wt) CaCL solution was drawn into a syringe, and the contents of the cryotube (thawed cells in cryopreservation solution) were withdrawn into the same syringe. Immediately thereafter, 1/3 of the contents of the syringe was injected into one chamber through the corresponding ports and valves of Examples 1 and 2. The other third was injected into the second chamber and the third third into the third chamber.

Example 4A: Washing DMSO from Cryopreserved Cells

This procedure was carried out in the chambers according to Example 1. The syringe was connected through the valve V3, the three-way stopcock was set to the "filter closed" position, the suspension was injected into container A of the chamber, the syringe was disconnected, the valve V3 was closed and the three-way stopcock was set to "filter open" position. A premoistened Hyalofast carrier was placed in all containers A. 330 thousand were used for each sample. cells. Since the gel starts to harden in approx. 60 seconds after combining all the components, the liquid with the cells must be applied to the moistened carrier immediately after mixing all the components. The liquid with the cells was absorbed into the carrier and within one minute the gelling reaction began. All chambers were placed in a 37°C incubator for 10 minutes. After the formation of the hydrogel, which serves to anchor the cells in the carrier, Ringer's solution, preheated to 37 °C, was transferred to a sterile syringe and 2 ml of this solution was carefully introduced into the container A of the chamber through valve VI, which is connected to the inlet/outlet port P1 to dilute the DMSO. After one minute, the solution was aspirated from the chamber with another sterile syringe through valve V2. Washing was repeated 2 more times. After washing, the therapeutic preparations consisting of cells, carrier and hydrogel were removed from the chamber and used to detect cell viability. The therapeutic preparations were removed with sterile tweezers through the hole O after unscrewing the screw cap U.

Example 4B: Washing DMSO from Cryopreserved Cells

This procedure was carried out in the chambers of Example 2. The chambers with the wetted carrier N inserted were rotated to a vertical position with the inlet/outlet ports P3 and P4 facing down. The carrier N was Chondrotissue®. The three-way taps K2, K3, K4 connected to the inlet/outlet ports P2, P3, P4 were set to the "filter open" position, while the three-way valve connected to the inlet/outlet port Pl was set to the "filter closed" position. The syringe containing the cell suspension was connected to valve VI, which is connected to the inlet/outlet port P1, and the suspension was injected into container A onto the carrier N. After injection of the suspension, the chamber was rotated to a horizontal position and gently rocked from side to side to spread the suspension on the surface of the carrier. Then the syringe was disconnected and all three-way taps Kl, K2, K3, K4 were set to the "filter closed" position. 330 thousand were used for each sample. cells. Each sample was mounted in one chamber.

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To improve the homogeneity and efficiency of carrier seeding, it is possible to repeat the procedure of deploying cells from other valves.

All chambers were placed in a 37°C incubator for 10 minutes. After 10 minutes, when it was formed by the conversion of fibrinogen contained in the platelet lysate into a fibrin hydrogel, which serves to anchor the cells in the carrier, the Ringer's solution warmed to 37 °C was transferred to a sterile syringe and 2 ml of this solution was carefully injected into container A through the valve VI connected to the PI input/output port. to dilute the DMSO, with all three taps K1, K2, K3, K4 set to "filter closed". After one minute, the solution was aspirated from the chamber with another sterile syringe through valve V4 connected to inlet/outlet port P4. Washing was repeated 2 more times.

After washing, the therapeutic preparations, the constructs formed by the cells, the carrier and the hydrogel were removed from the chamber and used for the detection of cell viability. The therapeutic preparations were removed from the chamber with sterile tweezers after removing the clamps D and lid B.

Example 5: Detection of cell viability after DMSO washing

For the detection of cell viability, the therapeutic preparations consisting of cells, carrier and hydrogel of Examples 4A and 4B were removed from the chamber of Example 1 through hole O after removing the upper screw cap U or from the chamber of Example 2 by removing the clamps D and opening the lid B, and stained with LIVE/DEAD® Viability/Cytotoxicity kit (according to the manufacturer's instructions), which contains Calcein AM dyes, which are among the best indicators of living cells, and Ethidium homodimer-1, which stains dead cells with damaged membrane integrity. After staining and rinsing, the stained therapeutic preparations were analyzed under a microscope. Image analysis was performed using the ImageJ program and the percentage of live and dead cells was calculated.

At the same time, the LIVE/DEAD® Viability/Cytotoxicity kit was used to stain cells that were frozen according to Example 3 A and thawed according to Example 4. After thawing, cells with cryopreservation medium were resuspended in 4 ml of Ringer's solution, centrifuged for 5 minutes at 300 rcf (relative centrifugation forces), the supernatant with DMSO was removed and the pellet resuspended in 1 ml of Ringer's solution. The measured values were used as control values.

TABLE 2 Percentage of live cells after DMSO washing from cryopreserved cells

Chamber according to Example 1 Chamber according to Example 2 Centrifugation Percentage of living cells 91 ±6 87 ±3 88 ±7

The results shown in Table 2 confirm that washing DMSO from cryopreserved cells according to Examples 4A and 4B is at least comparable to the conventional washing method by centrifugation.

Example 6: Deployment of freshly harvested cells into a biomaterial carrier

Passage 3 human WJ-MSCs were harvested using trypsin/EDTA enzyme and centrifuged (5 min, 1100 rpm) after addition of fetal bovine serum and saline. One million cells were resuspended in one milliliter of blood plasma, drawn into a sterile tube that already contained 25 µl of a 10% (wt.) CaCL solution. The cells were introduced into container A of the chamber according to Example 1. Three chambers were seeded from one syringe. A total of six chambers were fitted for each type of carrier. The support was cut commercial support (5 x 5 mm) hyalofast and cut commercial support (5 x 5 mm) chondrotissue. The carriers were moistened with the complete culture medium for 30 min before introducing the cells.

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Example 7: Cultivation of therapeutic preparations in the chamber according to Example 1

After seeding the cells according to Example 6, 2 ml of the complete culture medium, the same as in Example 3A, was injected into container A of the chamber according to Example 1 via the valve V2 connected to the input/output port P2 with a syringe. Then the three-way cock K was set to the "filter open" position, which allows air exchange in the system. For static cell culture, all seeded chambers were placed in a 37°C, 5% CO2 incubator. Air exchange took place through a 0.22 pm filter.

On the first, fourth, and seventh days, the 3D cell constructs with biomaterial carriers were removed from the chamber and cell viability was measured using the resazurin assay of Example 3A. The therapeutic preparations were then returned to the culture chamber, Ix washed in complete culture medium and further cultured in culture medium in a controlled atmosphere thermostat at 37°C, 5% CO2. Therapeutic preparations were removed and returned from/to container A with sterile tweezers through opening O after opening the screw cap U.

Flushing with complete culture medium was performed as follows: a sterile 5 ml syringe was connected to valve V2 connected to the inlet/outlet port P2 and the medium was aspirated from the system, the syringe was disconnected and the valve was closed. Using another 5 mL syringe, 2 mL of fresh medium was added through valve V3. Then the three-way tap K was set to the "filter open" position.

The results of the experiments show that the cells anchored in the material are vital and do not lose the ability to proliferate after two days of cultivation (Fig. 4). Cells cultured on the HF carrier retain their proliferative ability even on the seventh day of culture.

Example 8: Storage of cells in therapeutic preparations in a closed chamber for 2 to 6 days at 25 °C

In this example, only the hyalofast carrier (5x5 mm) was used for cultivation, which was fitted into both types of chambers (according to Example 1 and according to Example 2). 6 chambers of each type were set up for the experiment.

The trypsinized cells were seeded into vessel A of the chamber of Example 1 by the procedure of Example 6, and after the gelling reaction had taken place, 2 ml of complete culture medium, the same as in Example 3A, was injected via a syringe through the valve V2 connected to the inlet/outlet port P2. Then the three-way cock K was set to the "filter open" position, which allows air exchange in the system. For static cell culture, the chamber was placed in a controlled atmosphere incubator at 37 °C, 5% CO2. Air exchange took place through a 0.22 pm filter.

Trypsinized cells according to Example 6 were seeded into the container of chamber No. 2 according to Example 4B. After the gelling reaction had taken place, 2 ml of complete culture medium, the same as in Example 3A, was injected through the valve VI which is connected to the inlet/outlet port P1. The three-way taps K2, K3, K4 on the inlet/outlet ports P2, P3, P4 have been set to the "filter open" position, allowing air exchange in the system. For static cell culture, the chamber was placed in a controlled atmosphere incubator at 37 °C, 5% CO2. Air exchange took place through a 0.22 pm filter.

On the first, fourth and seventh days, the medium was changed in the chambers. The exchange of the culture medium was carried out according to the following procedure: in the chamber according to Example 1, a sterile 5 ml syringe was connected to the valve V2 connected to the input/output port P2 and the medium was aspirated from the system, the syringe was disconnected and the valve V2 was closed. Using another 5 mL syringe, 2 mL of fresh medium was added through valve V3. Then the three-way tap K was set to the "filter open" position. In the chamber of Example 2, a sterile 5 ml syringe was connected to the valve V3 connected to the inlet/outlet port P3 and the medium was aspirated from the system, the syringe was disconnected and the valve V3 was closed. Then the three-way tap K was set to the "filter open" position. Using another

-9CZ 36430 UI ml syringe, fresh medium was introduced into the container of the chamber using valve VI. connected to the input/output port Pl. The three-way taps K2 and K4 at the input/output ports P2 and P4 were set to the "filter open" position, while the three-way taps KI and K2 at the input/output ports P1 and P2 were set to the "filter closed" position.

On the eighth day, the cells in the therapeutic preparations were subjected to the resazurin test according to Example 3A. After the resazurin test, the therapeutic preparations were washed with complete culture medium and returned to the chambers with fresh medium. After one hour of culture in a 5% CO2, 37°C controlled atmosphere thermostat, the chambers were removed from the controlled atmosphere thermostat, hermetically sealed to exclude air, and placed in a constant 25°C thermostat for 2 or 6 days . Cultivation for one hour prior to removal from the controlled atmosphere thermostat sufficiently saturated the culture medium with carbon dioxide. Neither the chamber nor the therapeutic agent was manipulated during the entire storage period of the cells at 25 °C. After 2 or After 6 days, the therapeutic preparations according to Examples 4A and 4B were removed from the chamber vessels, and a resazurin test was performed according to the procedure according to Example 3A to determine the viability/survival rate of the cells after storage 2, respectively. 6 days in non-physiological conditions for the cells. At the same time, the pH of the medium in which the cells were stored for 2 or 6 days. The ideal pH value of the medium for cells is 7.2 to 7.4. Values below 6.8 or above 8.2 are unsuitable for cells and may cause cell death. The pH of the medium, which was taken on the 8th day of cultivation before the resazurin test, was also measured.

The pH of the medium in which the cells were stored for 2 or 6 days, did not significantly change compared to the pH of the control medium in which the cells were cultured in an optimal thermostat environment with a controlled atmosphere and temperature (Table 3). The resazurin test showed that the cells in the therapeutic preparations maintain a high rate of survival after two and six days of storage in a closed chamber without access to air and gases at a temperature of 25 °C, are vital and able to proliferate further (Fig. 5). A period of 2 to 6 days of storage without further manipulation, in a sterile environment, is necessary to perform the safety tests required for the release of the therapeutic product and for its transport to the site of application.

Table 3 - pH media before and after 2 and 2 days of storage in a closed chamber

pH of the medium day 0 day 2 day 6 Chamber according to Ex. 1 7.21 ±0.23 7.54 ± 0.27 7.63 ± 0.26 Chamber according to Ex. 2 7.32 ±0.15 7.57 ±0.18 7.67 ± 0.22

Claims (7)

PROTECTION CLAIMS 1. A chamber for the preparation of a therapeutic preparation, wherein this chamber contains at least one container (A), characterized in that the container (A) is provided with at least two input/output ports (P1), (P2), (P3), (P4) ) adjusted for aseptic introduction and withdrawal of fluids, while at least one of the inlet/outlet ports (P1), (P2), (P3), (P4) is provided with a three-way cock (K), (KI), (K2), ( K3), (K4), one supply of which is equipped with a filter (F), (Fl), (F2), (F3), F4) for cleaning the incoming gases and the other supply with a closable valve (VI), (V2), (V3 ), (V4), and while the other inlet/outlet ports (Pl), (P2), (P3), (P4) may optionally also be provided with valves (VI), (V2), (V3), (V4), while the container (A) is provided with at least one closable opening (O) for introducing and removing the carrier (N). 2. Chamber according to claim 1, characterized in that there are two to six, preferably two to four, input/output ports (P1), (P2), (P3), (P4). 3. Chamber according to claim 1 or 2, characterized in that the closable opening (O) for introducing and removing the carrier (N) is equipped with a screw cap (U), which can optionally be further equipped with a three-way tap (K) with a filter (F ) and/or valve (V). 4. Chamber according to claim 1 or 2, characterized in that the closable opening (O) for introducing and removing the carrier is provided with a sealing lid (B). 5. Chamber according to any one of claims 1 to 4, characterized in that it is further provided with an inlet and an outlet connected to a peristaltic or injection pump. 6. A kit for the preparation of a therapeutic preparation, characterized in that it contains a chamber according to any one of claims 1 to 5, and a container with a 1 to 10% (mass.) aqueous solution of CaCF. 7. The kit according to claim 6, characterized in that it further contains at least one other component selected from: - carrier, preferably located in the container (A) of the chamber; - containers with platelet lysate or blood plasma; - containers with dimethyl sulfoxide; - containers with 0.9% (mass.) NaCl aqueous solution or Ringer's solution.