Method of preparation of autologous at-mscs cells

Abstract

A method for the preparation of human autologous AT-MSC cells that have high viability and functional potential of MSC cells and are free of contamination by other cells is disclosed. The cells obtained by the method of the invention can be used for therapeutic purposes in clinical practice.

Description

[0001] Method of preparation of autologous AT-MSCs cells

[0002] The subject of the invention is an improved process for preparation of autologous mesenchymal stem / stromal cells from adipose tissue (AT-MSCs). The cells obtained by the method of the invention can be used for treatment in clinical practice, under the name MesoCellA.

[0003] Patent application US2018117088 describes a method for preparing AT-MSCs cells from adipose tissue.

[0004] The aim of the invention is to provide an improved process for preparing autologous AT-MSC cells with a viability of at least 95%. Another objective of the invention is also to provide a formulation that is substantially free of contamination by cells other than mesenchymal stem / stromal cells (MSCs), such as erythrocytes (RBCs), leukocytes or pericytes. In addition, it is desirable to prepare AT-MSCs having the morphology, phenotype, trilinear differentiation potential characteristic of MSCs. In particular, the prepared cells should have, among others, the following antigens on their surface: CD44, CD73, CD90, CD 105 (characteristic of MSCs) and at the same time not to present, among others, the following antigens on their surface: CD45, CD34, CD14, CD19 and HLA-DR antigen, as well as demonstrate the ability to trilinear mesenchymal differentiation (to osteoblasts, chondroblasts and adipocytes).

[0005] Due to the intended use of the manufactured cells, which are used to prepare medicinal products, it is necessary to develop a method that will ensure the standardization of the manufactured formulation, in particular to obtain AT-MSCs with repeatable and comparable biological properties.

[0006] The subject of the invention is defined in details in the appended claims.

[0007] Exemplary embodiments of the invention are further illustrated in the accompanying drawings.

[0008] Fig. 1 shows a simplified diagram of an exemplary embodiment of the method of the invention.

[0009] Fig. 2 shows optimization of isolation of the stromal-vascular fraction (SVF) from the adipose tissue. Washing the adipose tissue (collected during liposuction) with a solution of a salt which is isotonic to human cells, including for example PBS, with the addition of an Antibiotic / Antimycotic solution - comprising antibiotics and an antifungal agent (penicillin, streptomycin and amphotericin B) does not cause the loss of adherent cells of the SVF fraction (PBS after washing the adipose tissue was centrifuged, and then the sedimented pellet comprising RBCs and tissue fragments was resuspended in culture medium and cultured for 9 days; growth of only single adherent cells per microscopic field was observed, which confirmed that the adipose tissue washing cycle with saline solution, including PBS, does not cause the loss of adherent fraction of SVF cells) and significantly reduces the amount of red blood cells in the remaining adipose tissue.

[0010] Fig. 3 shows optimization of isolation of SVF cells. Examination of impact of the additional step of RBCs lysis on the efficiency of SVF isolation. The effect of RBCs lysis on the efficiency of SVF isolation is shown as (A) the number of isolated SVF cells per fat mass unit; (B) viability of isolated SVF cells (%). Lysis of RBCs was performed using a dedicated lysis buffer (comprising ammonium chloride solution). SVF cells were incubated with lysis buffer for 5 or 10 minutes, then SVF cells were counted and their viability was determined. (C) Qualitative evaluation of adhesion capacity of isolated SVF cells prepared on the basis of microscopic observation of the derived adherent cell culture conducted prior to the first cell passage.

[0011] Fig. 4 shows optimization of isolation of SVF cells. (A) Examination of the efficiency of SVF cell isolation for selected collagenase activities. The adipose tissue was incubated with 0.1 , 0.2 or 0.3 PZ U / ml collagenase for 40 minutes at 370C. The isolation efficiency of SVF cells per 1 g of lipoaspirate is shown in the graph, while the viability of the isolated cells is shown in the table below. (B) The ability of the isolated cells to undertake active growth in in vitro culture for the used enzymatic activities was evaluated by light microscopy and shown in the pictures. (C) Examination of adipose tissue digestion time. The adipose tissue was incubated with collagenase having enzyme activity of: I) 0.1 ; ii) 0.2; ill) 0.3 PZ U / ml for 60 minutes at 37 ° C. The isolation efficiency of SVF cells was shown as the total number of SVF cells obtained per 1 g of lipoaspirate and compared with the isolation efficiency obtained after incubation of the adipose tissue with collagenase having enzyme activity of 0.2 PZ U / ml for 40 minutes at 370C.

[0012] Fig. 5 shows characteristics of isolated AT-MSC cells according to the criteria defining multipotent MSCs cells proposed by the International Society for Cell and Gene Therapy (ISCT). (A) Morphology of native AT-MSCs cells by light microscopy & Analysis of the multipotential potential of AT-MSCs in in vitro culture performed by specific histological staining. AT-MSCs were cultured in appropriate differentiation media: in case of differentiation towards osteoblasts / osteocytes - StemPro Osteogenesis Differentiation Kit (for 21 days), in case of differentiation towards chondroblasts / chondrocytes - StemPro Chondrogenesis Differentiation Kit (for 21 days) or StemPro Adipogenesis Differentiation Kit (for 12 days) in case of differentiation towards fat cells. After 12 or 21 days of differentiation, AT-MSCs cells were fixed with paraformaldehyde and stained with: alizarin red S (the red color of calcium phosphate deposits is characteristic of osteogenic differentiation), alcian blue (the blue color of sulfated proteoglycans is characteristic of chondrogenic differentiation) or left unstained to observe the deposition of lipoprotein droplets in the cytoplasm. Scale: 50 pm. (B) Representative histograms of negative (CD14, CD19, CD45, CD34, CD31 , KDR) and positive (CD44, CD90, CD74, CD105) expression markers for MSC cells, as well as HLA-DR antigen on the surface of AT-MSCs cells. The analysis was performed using flow cytometry.

[0013] Fig. 6 shows results of stability test of the obtained AT-MSC cells resuspended in a carrier solution. (A) Viability of 20±2x106AT-MSCs suspended in 1.5 ml of 0.9% NaCI comprising 10% human albumin solution (20% human albumin solution diluted in a 1 :1 ratio with 0.9% NaCI solution) and for comparison, as a reference solution, aMEM comprising 2 lU / ml heparin and 5% MultiPL'1 OOi (which is not dedicated for use in humans), at a temperature of 2-8 ° C. (B) Viability of 20±2x106AT-MSCs resuspended in 1.5 ml of 0.9% NaCI supplemented with 10% human albumin solution (20% human albumin solution diluted in a 1 :1 ratio with 0.9% NaCI solution) at a temperature of 2-80C. Human albumin solutions (HSA) used to prepare the carrier solution were manufactured by Grifols (HSAGrifols) or CSL Behring (HSA Behring). The test was conducted for 3 cell-based products.

[0014] Fig. 7 shows stability of the obtained cells. Viability of 20±2x106AT-MSCs suspended in a 20% human albumin solution diluted in a 1 :1 ratio with 0.9% sodium chloride solution (corresponding to a final 10% concentration of human albumin solution) at 2-8°C (A) or at room temperature (RT) (B). The viability was assessed using a microscope and trypan blue (Ph. Eur. 2.7.29). The test was carried out in a manufacturing site (Biological Advanced Therapy Medicinal Products (ATMP) Manufacturing Facility).

[0015] Fig. 8 shows adhesion of AT-MSCs to the surface of culture vessels during in vitro culture. AT- MSCs were collected during the stability test at the following time points of 0 (immediately after the start of the test), as well as 4, 8, 12 and 24 hours (h) after the start of the test, and then the cells were seeded on culture vessels. Images of the cells were taken exactly 72 hours from the moment of seeding the cells on the culture vessels.

[0016] Detailed description of the invention

[0017] The experimental work that resulted in the method according to the invention allowed to identify its features that are crucial for the quality and viability of the obtained AT-MSCs cell formulation. The unique properties of the formulation obtained according to the invention are shown in Example 2.

[0018] In an exemplary embodiment, the method of the invention generally comprises the following steps:

[0019] 1. Isolation of the stromal-vascular fraction (SVF) cells from the adipose tissue and establishment of primary culture of human mesenchymal stem / stromal cells (AT- MSCs),

[0020] 2. AT-MSCs cell culture consisting of cell passage cycles and medium replacement in cell culture,

[0021] 3. Preparing of the final product and its release.

[0022] The inventors identified several key elements of this method that have a significant impact on the quality and viability of the final AT-MSC cells.

[0023] In the first step of the method according to the invention, isolation of the SVF fraction is carried out and the primary culture of AT-MSC cells is established.

[0024] In an exemplary embodiment of the method of the invention, the lipoaspirate is preferably washed with a buffered saline solution (PBS) comprising a mixture of antibiotics and an antifungal agent (2X concentrated Antibiotic-Antimycotic (Anti / Anti) solution, which includes: penicillin, streptomycin and amphotericin B. In a preferred embodiment, the lipoaspirate is diluted in a 1 :1 volume ratio with a saline solution (PBS) comprising 2x concentrated Anti / Anti solution, the number of tissue rinses: 3-6, the washing is carried out until red color disappears. The purpose of this step is to reduce the content of red blood cells (RBCs) and reduce any bacterial / fungal contamination. It is acceptable to perform this step using another solution isotonic to human cells such as culture medium, sodium chloride (NaCI) solution, Ringer's solution, as well as an isotonic solution without the addition of antibiotics, provided that the source tissue for AT-MSCs isolation is collected aseptically and cell isolation and culture process is also performed aseptically.

[0025] In known solutions, the lysis of RBCs is most commonly performed using a suitable solution comprising ammonium chloride solution (the so-called Lysing Buffer). The rinsing of the lipoaspirate with a solution isotonic to human cells, preferably a saline solution (PBS) comprising a mixture of antibiotics and an antifungal agent, carried out in accordance with the invention, is an alternative and effective method of removing RBCs from the lipoaspirate that is safe for nucleated cells. In addition, washing the tissue with saline solution (PBS) or another solution isotonic to human cells also removes bacteria / fungi that may appear accidentally during the tissue collection procedure. The addition of a mixture of antibiotics and an antifungal agent to a saline solution (PBS) in the first isolation step significantly increases the likelihood of obtaining a sterile cell culture and preparing a sterile final cell product.

[0026] In the experimental work that led to the invention, the inventors unexpectedly observed that washing lipoaspirate with saline solution (PBS) significantly reduces the content of RBCs and does not cause the loss of adherent cells (which was demonstrated by the inventors experimentally: the saline solution (PBS) was centrifuged after washing adipose tissue, and then the pellet containing RBCs and tissue pieces was resuspended in culture medium and cultured for 9 days; no growth of adherent cell fraction was observed, Fig. 2).

[0027] In addition, the inventors compared the efficiency of SVF isolation using the protocol of the invention (comprising the step of washing the lipoaspirate using a saline solution (PBS) comprising a mixture of antibiotics and an antifungal agent) with the efficiency obtained using another known isolation protocol, in which isolated SVF cells were additionally incubated in a hypotonic lysis buffer to reduce the number of RBCs.

[0028] The highest number of isolated SVF cells, which at the same time showed the highest viability, were obtained for the SVF isolation protocol of the invention (comprising the step of washing the lipoaspirate using a saline solution (PBS) comprising a mixture of antibiotics and an antifungal agent) compared to the alternative protocols comprising the step of lysis of RBCs using a dedicated lysis buffer (Fig. 2 A, B). Furthermore, the performed microscopic observations confirmed a higher adhesion capacity to the culture surface of the cells isolated by omitting the lysis step of RBCs using the lysis buffer (Fig. 2C).

[0029] In a further step of the method of the invention, the adipose tissue is subjected to enzymatic digestion by collagenase,, preferably of GMP grade. The use of collagenase with comparable properties and quality, including R&D or cGMP grade collagenase, is also acceptable.

[0030] In an exemplary embodiment of the process of the invention, the adipose tissue is digested with collagenase having the final optimized enzymatic activity of 0.2 PZ U / ml. The temperature and digestion time are - 37±1 ° C, 35-40 min, respectively. It is possible to perform this step at a different temperature ensuring enzyme activity, such as 30-40 °C, however, the isolation efficiency and cell viability may be lower. The used collagenase is a mixture of, among others, class I and II collagenases. After digestion, collagenase activity is reduced by cooling down the test tubes with digested tissue - by centrifugation at 17 ± 1 ° C. It is also acceptable to reduce collagenase activity by diluting it, i.e. adding a solution isotonic to human cells to the test tubes with the digested tissue. As a result, the method of the invention uses an effective, safe, repeatable, relatively short step of isolation of SVF cells.

[0031] In known methods, the digestion of the adipose tissue and other tissues is most often carried out using a selected concentration (by percentage, molar, by weight) of collagenase. Since each batch of collagenase has a different enzymatic activity, the method of the invention uses enzyme activity parameter instead of collagenase concentration. This allows for better standardization of the digestion step and reduces differences between subsequent cell isolations, making the isolation protocol universal and standardized. The temperature range at which digestion is carried out has been specified in accordance with GMP requirements and it allows maximum collagenase activity to be maintained (according to the principles of enzymology and to the best of our knowledge). It was also experimentally confirmed that prolonged digestion of adipose tissue with collagenase does not increase the efficiency of SVF cell isolation.

[0032] In a preferred embodiment, a collagenase of the GMP grade was used, which is a mixture of, among others, class I and II collagenases. Typically, one type (class) of collagenase is used in known methods.

[0033] In the work that led to the invention, the authors conducted an assessment of the enzymatic activity of collagenase suitable for the isolation of SVF cells - the following enzymatic activities of collagenase were tested during the optimization of the SVF isolation procedure: 0.1 , 0.2, 0.3 PZ U / ml. All tested collagenase activities allow to effectively isolate SVF cells with a viability equal to or higher than 95%. However, the enzymatic activity of 0.2 PZ U / ml allows to obtain a primary culture with a higher proliferative capacity compared to other collagenase activities tested (Fig. 3B). The selected enzymatic activity (0.2 PZ U / ml) was used in the particularly preferred embodiment of the SVF cell isolation protocol.

[0034] It was also observed that extending the enzymatic digestion time to 60 min does not increase the isolation efficiency of SVF cell fraction (Fig. 4D).

[0035] In a further step of the method of the invention, AT-MSCs cell culture is carried out, consisting of cell passage and medium replacement cycles.

[0036] In an exemplary preferred embodiment of the method of the invention, isolated SVF cells constituting a heterogeneous cell population, including AT-MSCs, are cultured from passage 3 to passage 5 (P3-5) before being used in clinical practice.

[0037] In a particularly preferred embodiment, AT-MSCs are cultured in an aMEM medium supplemented with 5-10% platelet lysate (human platelet lysate (hPL); the used medium is intended for MSC culture for clinical applications), 2 lU / ml heparin, and a 1X concentrated Antibiotic-Antimycotic solution (a solution of penicillin, streptomycin, and amphotericin B is used only until the first passage). It is also allowed to conduct cell culture in a different culture medium dedicated for human cells, including free of antibiotics and antifungal agent. In such particular case, the source tissue for AT-MSC isolation is collected aseptically and the isolation process and cell culture are also performed aseptically. It is also acceptable to use a plate lysate that does not require the addition of heparin to prepare the medium.

[0038] SVF cells, and then AT-MSCs cells, are cultured at 35-40°C, preferably 37°C, under air with CO2 content enriched to 5% and O2 in the range of 0.5-23%, preferably 21%.

[0039] In a particularly preferred embodiment, all materials and reagents used for the production of AT-MSCs are sterile and of GMP or equivalent grade (as well as coming from qualified suppliers / manufacturers), which allows the use of the manufactured cells in clinical practice. The use of materials and reagents of the R&D grade is acceptable, provided that their appropriate quality is demonstrated, including sterility, biocompatibility for human cells, absence of pyrogens and ensuring the growth of AT-MSC cells.

[0040] The culture conditions proposed in accordance with the invention (preferably also in accordance with the requirements of GMP) allow to obtain AT-MSCs having the morphology, phenotype, trilinear differentiation potential characteristic of MSCs. AT-MSCs also exhibit high viability and genetic stability, what confirm their safety (see also Fig. 7, Table 8).

[0041] Furthermore, the established preferred culture conditions are suitable for MSCs cultures, including in particular human MSCs for clinical applications. The use of a solution of antibiotics and an antifungal agent in the first days of culture (until the first passage) excludes the possibility of a microbial contamination that could occur during the adipose tissue collection procedure. Thanks to this, the inventors are able to obtain a sterile final product.

[0042] In the work that led to the invention, the authors decided not to use fetal bovine serum (FBS) as a source of nutrients for cells. FBS is commonly used in prior art cell cultures (also under GMP conditions). Instead of FBS, the inventors used human plate lysate (hPL), which provides nutrients to growing cells. During AT-MSCs culture using culture medium supplemented with FBS, reduced cell proliferation capacity was observed along with larger cell size, whereas AT- MSCs cultured in hPL-supplemented media unexpectedly showed more effective proliferation capacity and maintained a smaller size. Cell proliferation efficiency is a critical parameter during the manufacture of cell-based medicinal products, which determines whether researchers will be able to obtain a sufficient number of cells to manufacture a medicinal product based on them. In addition, replacing FBS with hPL eliminates the potential risk of transmission of prions of a dangerous disease - bovine spongiform encephalopathy, and other potential zoonoses. In the work that led to the invention, the inventors experimentally excluded the need to coat the surface of culture vessels with extracellular matrix (ECM) proteins in order to obtain optimal cell growth, such as fibronectin, which is commonly used in the prior art, as demonstrated in the present embodiment - being ineffective. Unexpectedly, it was found that the use of hPL eliminates the need for additional coating of the culture surface with ECM proteins and effectively promotes the adhesion of AT-MSCs to the plastic surfaces of the culture vessels.

[0043] The work that led to the invention also determined the optimal concentrations of antibiotics and the duration of their use, which guarantee the sterility of AT-MSCs culture, as well as the possibility of their effective removal, and the absence of antibiotics in the final product.

[0044] It was also found that the use of hPL-enriched culture medium allows to obtain a higher proliferative potential of AT-MSCs compared to cells grown in FBS-enriched media (Table 1). In addition, the need to coat the surface of culture vessels with fibronectin was experimentally excluded (Table 2), because this step, recommended in the prior art, does not actually increase the efficiency of cell culture and constitutes an additional manipulation increasing the risk of contamination in cell culture. In addition, the use of 1X concentrated (i.e. 1%) solution of penicillin, streptomycin and amphotericin B, corresponding respectively to the following concentrations: 100 U / mL penicillin, 100 pg / mL streptomycin, and 0.25 pg / mL amphotericin B) until the first passage (i.e., preferably 4-5 ± 2 culture days) allows to obtain a sterile culture and the manufacturing of a cell-based final product (Table 3).

[0045] In the proposed preferred embodiment of the invention, the identity of the isolated and cultured cells as MSCs was confirmed under the described culture conditions. The identity of the cultured cells as MSCs was confirmed based on the relevant phenotype assessed according to specific criteria published by the International Society for Cell and Gene Therapy (Dominici et al., 2006). The isolated and expanded adherent fraction of AT-MSCs maintained under standard culture conditions (described above) are elongated, spindle-shaped cells with morphology similar to fibroblasts exhibiting adhesion to polystyrene surfaces of culture vessels, having on their surface, among others, the following antigens: CD44, CD73, CD90, CD105 (characteristic of MSC cells) and with the simultaneous lack of, among others, CD45, CD34, CD14, CD19, CD31 , KDR and HLA-DR antigens, and showing the ability to trilinear mesenchymal differentiation (to osteoblasts / osteocytes, chondroblasts / chondrocytes and adipocytes) (Fig. 4).

[0046] Table 1. Efficiency of AT-MSCs proliferation cultured in a dedicated basal medium supplemented with: i) 10% FBS; ii) 10% hPL. Efficiency of cell cultures 1

[0047]

[0048] Table 2. Efficiency of AT-MSCs proliferation cultured in medium supplemented with 10% FBS; or 10% hPL on: i) culture vessels coated with fibronectin; ii) culture vessels not coated with fibronectin.

[0049] Table 3. Sterility of cell culture and final product comprising AT-MSCs.

[0050] In a further step of the method of the invention, the final product is prepared and released.

[0051] In an exemplary preferred embodiment of the method of the invention, AT-MSCs are collected during one passage that falls between passage 3 and passage 5 (P3-5), washed with saline solution (PBS), mixed with a carrier solution and packaged in direct container. It is acceptable to wash cells with another solution isotonic to human cells and to collect cells in different time interval of culture (i.e. between other passages), provided that MSCs cells with high viability and with a stable phenotype, genotype and biological properties characteristic of MSCs are obtained.

[0052] In the work that led to the invention, the inventors unexpectedly determined that the suspension of AT-MSC cells in 0.9% sodium chloride solution comprising 10% human albumin solution (20% human albumin solution diluted in a 1 :1 ratio with 0.9% NaCI) allows the high viability of these cells to be maintained, which is crucial for their optimal biological properties (to the best of our knowledge).

[0053] It has also been observed that the viability of AT-MSCs suspended in NaCI solution supplemented with 10% human albumin solution (HSA, 20% human albumin solution diluted 1 :1 with 0.9% NaCI) is high and comparable to the viability of these cells resuspended in standard culture medium (intended for culturing human MSCs cells for clinical applications, but not intended for direct administration into humans). No significant difference in the viability of AT-MSCs was observed at the analyzed time points after product formulation (Fig. 6A), which confirms the proper selection of the carrier solution for product formulation. In addition, the comparable high viability of AT-MSCs resuspended in 0.9% NaCI solution supplemented with 10% human albumin solution from CSL Behring supplier and another manufacturer - Grifols (Fig. 6B) has also been confirmed. It is also acceptable to resuspend AT-MSC cells in a different solution isotonic to human cells, such as, among others: culture medium, NaCI solution, Ringer's solution and with a different concentration of albumin or other solution comprising stabilizing proteins, provided that high viability, stable phenotype, genotype and biological properties of cells characteristic of MSCs are maintained.

[0054] Example 1 . Preparation of autologous AT-MSCs

[0055] A schematic diagram of an exemplary embodiment of the method of the invention is shown in Fig. 1 , while preferred embodiments of its successive steps are discussed in the example described below.

[0056] Isolation of SVF cells and establishment of primary culture

[0057] Transfer the collected adipose tissue to centrifuge tubes (falcon type) and then wait a few minutes until the infiltration fluid separates from the adipose tissue (adipose tissue - upper phase). This step can also be done using a centrifuge. To do this, place the tubes comprising the tissue in the device and then accelerate the centrifuge to 200xg. After the phases are separated, using a serological pipette remove the infiltration fluid from each falcon tube, then add the saline solution (PBS) with Anti-Anti (comprising 2X concentrated solution of antibiotics and an antifungal agent - Antibiotic-Antimycotic: penicillin 200 U / ml, streptomycin 200 pg / ml and amphotericin B 0.5 pg / ml) with a volume equal to the volume of tissue in the falcon tube. Screw the cap on each falcon tube and then mix the content of tubes by inverting them several times. Wait a few minutes until the phases separate. This step can also be done using a centrifuge (see above). Open the test tubes with the tissue and then, using serological pipettes, remove the saline solution (PBS) with the Anti-Anti (lower phase) in each test tube as much as possible, leaving adipose tissue in the falcon tubes. Repeat washing twice. In case the fluid is still strongly colored with erythrocytes, perform additional washes (up to 3 consecutive washing cycles).

[0058] Remove the saline solution (PBS) with the Anti-Anti solution from the last lipoaspirate rinse. To each falcon tube comprising adipose tissue, add a volume of collagenase solution using a serological pipette to achieve a final enzyme activity of 0.2 PZ U / ml. Close the falcon tubes and then incubate them in a shaker oven at 200 rpm at 37°C ± 1°C for 35-40 minutes. After incubation, visually assess whether the adipose tissue has been digested. Then spin the falcon tubes at 370xg, 10 min, at 17 ± 1 °C. Using a serological pipette, remove the oily and aqueous phases so as to leave only the pellet. Perform the operation for each falcon tube. Then add saline solution (PBS) to each falcon tube and pipette the pellets. Prepare a new falcon tube of volume 50 ml. Apply a filter with a pore diameter of 100 pm to the falcon tube, onto which apply a suspension of cells. Rinse the tissue fragments remaining on the filter with saline solution (PBS). Fill the collective falcon tube comprising the isolated cells with saline solution (PBS) to a volume of 50 ml and spin at a speed of 350xg, 7 min at room temperature.

[0059] After centrifugation, remove the supernatant from the pellet and resuspend the pellet in saline solution (PBS). Then count the cells and determine their viability. Isolated cells should be evenly seeded onto culture flasks in Culture Medium-1 (i.e. aMEM containing 2 lU / mL heparin, 5-10% plate lysate, 1X concentrated Antibiotic-Antimycotic solution). Place the flasks in a culture incubator. Proceed with the culture at 37°C, in an atmosphere of 5% CO2.

[0060] Control of the primary culture establishment step and the first and subsequent replacement of the culture medium

[0061] Replace the culture medium 24-48 hours after establishment of the cell culture.. To do it, remove the entire volume of culture medium. Gently rinse the cells with saline solution (PBS). Using a serological pipette, add fresh Culture Medium-1. Conduct microscopic evaluation of cells. Then place the culture back in the incubator. Proceed with the culture at 37°C, in an atmosphere of 5% CO2.

[0062] End of primary culture - 1st passage

[0063] Perform the first passage 4-5±2 days after the primary culture is established. At this stage the following parameters should be assessed: a) symptoms of potential infection in the culture - based on the observation of color of culture medium and the appearance of turbidity of the culture medium, b) percentage of the area occupied by cells (confluence), c) cell morphology in culture. After macroscopic and microscopic observation, proceed to the cell passage. Pour out all of the culture medium and rinse the cells with 20 mL of saline solution (PBS) using a serological pipette. Pour out or remove the saline solution (PBS) using a serological pipette. Repeat rinsing twice. Then, using a serological pipette, add the Tryple Select solution to each bottle and distribute it evenly. Incubate culture flasks at 37°C. After approx. 2 minutes, gently hit the side of the flask and assess whether the cells have detached from the culture surface. Then add saline solution (PBS). Rinse culture surface and transfer the entire volume of the culture flask into the centrifuge tube. Spin the cells at 300xg, 5 min, at room temperature. Pour out or remove the supernatant using a serological pipette. Resuspend the pellet in saline solution (PBS). Then count the cells and determine their viability using trypan blue.

[0064] The establishment of secondary culture

[0065] In this step, counted cells should be seeded onto new culture vessels, such as e.g. flasks, for further culture in Culture Medium-2 (i.e. ctMEM comprising 2 ILI / mL heparin, 5-10% platelet lysate, no antibiotics and no antifungal agent added). Gently rock each bottle to evenly distribute the cell suspension into culture surface. Proceed with the culture at 37°C, in an atmosphere of 5% CO2.

[0066] The control and replacement of medium in secondary culture

[0067] Macroscopic and microscopic evaluation of AT-MSC cells in the culture should be carried out 24-48 hours after the secondary culture is established. If necessary, replace the medium. Gently pour out or remove all of the medium using a serological pipette. Using a serological pipette, add fresh Culture Medium-2. Then place each flask back in the incubator. Proceed with the culture at 37°C, in an atmosphere of 5% CO2 until the required confluence is achieved. When confluence is >70%, cell passage can be performed as described below.

[0068] End of secondary culture and further culture - 2nd and subsequent passages

[0069] Once >70% confluence is reached, perform cell passage. To do this, pour out all of the culture medium and rinse the cells with saline solution (PBS) using a serological pipette. Repeat rinsing twice. Then, using a serological pipette, add the Tryple Select solution to each flask and distribute it evenly into the culture surface. After approx. 2 minutes, gently hit the side of the flask and assess whether the cells have detached from the substrate. Then add saline solution (PBS) to each culture flask. Rinse them and transfer the entire contents of each culture bottle into the collective centrifuge tube. Spin the cells at 300xg, 5 min, at room temperature. Pour out or remove the supernatant using a serological pipette. Suspend the pellet in saline solution (PBS). Then count the cells using Burker’s chamber and determine their viability using trypan blue. If a sufficiently large number of cells is obtained (i.e. min. 1.5x106), seed them in a T500 flask (surface area of 500 cm2) in about 100 ml of Culture Medium-2 (per one T500 flask). Proceed with the culture at 37°C, in an atmosphere of 5% CO2. When the cells reach >70% confluence, passage them again in exactly the same way as described earlier. The culture should be carried out until the required number of cells is reached to allow the release of the cell-based product (preferably culture at 3-5 passage).

[0070] End of AT-MSCs cell culture (3-5 passage)

[0071] Conduct macroscopic and microscopic evaluation of AT-MSCs in the culture. For this purpose, the following parameters should be assessed: a) symptoms of potential infection in the cell culture, b) percentage of the area occupied by cells (confluence), c) cell morphology in culture.

[0072] After macroscopic and microscopic evaluation, pour out all of the medium from the culture vessels. Perform cell passage. To do this, rinse the bottles with saline solution (PBS). Pour out the saline solution (PBS). Then, using a serological pipette, add the Tryple Select solution to each vessel and distribute it evenly. After approx. 2 minutes, gently hit the side of the bottle and assess whether the cells have detached from the surface area. After the cells have detached from the culture surface, transfer the entire contents of the T500 flask into a centrifuge tube. Then rinse the flask additionally with saline solution (PBS) and transfer into the same centrifuge tube. Spin the cells at 300xg, 5 min, at room temperature. Pour out or remove the supernatant using a serological pipette. Pipette the pellets and transfer them to one collective centrifuge tube. Resuspend the cells in saline solution (PBS) and count them.

[0073] Acceptance criteria for the manufacture of the cell-based investigational product: a) cell number: no deviations: number of living cells sufficient for product preparation and quality control tests (according to the protocol of the specific clinical use of the manufactured product) result outside the specification: number of living cells insufficient for product preparation and quality control tests b) cell viability: no deviations: > 80% result outside the specification: <80%

[0074] Preparing of MesoCellA product Fill the falcon tube containing AT-MSC cells intended for the cell-based product with saline solution (PBS) to a volume of 50 ml and spin at 300xg, 5 min, at room temperature. After centrifugation, pour out or remove the supernatant using a pipette. Using an automatic pipette, resuspend the pellet in a specific volume of a solution, for example preferably in a solution isotonic to human cells containing 10% human albumin. Collect the specific volume of cell suspension (containing the appropriate number of live AT-MSCs cells) and transfer to the direct packaging of the product. Close the product package.

[0075] At the appropriate steps of culturing and preparing of the product, take samples for quality control tests.

[0076] Example 2. MesoCellA product stability test.

[0077] As the biological activity (including potency) of the cell-based product is linked to their viability and identity, these parameters were tested during stability tests carried out immediately after preparing of the final product and then after 4, 8, 12 and 24 hours of storage of the product under the following conditions: 1) 2-8°C (storage conditions); 2) room temperature (RT).

[0078] Stability tests were performed on 3 different validation series manufactured in accordance with Example 1 , designated as No.: 20210318 / WAL1 , 20210322 / 1 / WAL2, 20210322 / 2 / WAL3 manufactured in the ATMP Manufacturing Site (GMP-certified manufacturing facility).

[0079] Ensuring the durability (stability) of the product ensures that the patient receives a product with the highest quality features, which in turn translates into a therapeutic benefit for them.

[0080] Surprisingly, it was found that 20±2 x 106AT-MSC cells resuspended in 0.9% NaCI solution supplemented with 10% human albumin solution and stored at 2-8 °C showed high viability up to 24 hours after formulation. However, optimal biological properties measured as the ability to undertake active growth in in vitro culture are observed up to 12 hours after formulation. Although the cells are viable for up to 24 hours, they maintain optimal biological functions for up to 12 hours. Therefore, the stability time (at 2-8 ° C) is not 24 hours, but 12 hours. Many researchers estimate stability time solely based on cell viability or identity parameters. We have shown that the viability and identity parameters of cells are not sufficient to estimate their stability. The functionality of cells must be considered, which in our model has been demonstrated as the ability of cells to undertake active growth in in vitro culture.

[0081] It has been experimentally confirmed that the product is stable (AT-MSCs are viable and exhibit adequate phenotype) for 24 hours (h) at 2-80C and for 8 h when stored at room temperature (Fig. 6). The identity of the AT-MSCs before the formulation of the final product (0 h), as well as after 8 and 24 h after formulation, was confirmed in accordance with the parameter "Identity / phenotype" presented in Tables 4 and 5. Optimal biological properties of AT-MSCs measured as the ability to undertake active growth in in vitro culture are observed up to 12 h after product formulation (Fig. 7, Table 6).

[0082] On the basis of the conducted tests, the shelf life of the AT-MSCs cell suspension was experimentally determined to be 12 hours. The preferred storage temperature of the product is 2-8°C, however, stability tests have confirmed that it can only be stored in RT for up to 8 h.

[0083] Although the product specification is maintained up to 24h to ensure the highest viability and biological quality of the product, a shorter storage time is recommended before administration to the patient (12h). The type of used direct container does not affect the viability and identity of AT-MSCs, but may affect the functionality of these cells.

[0084] Table 4. Cell identity during storage stability tests at: 2-8° C.

[0085] Table 5. Cell identity during storage stability tests at: RT.

[0086] Table 6. Semi-quantitative assessment of the adhesion of AT-MSCs to the surface of culture vessels depending on the type of direct packaging for the product and the length of storage of the product in this packaging [developed on the basis of Fig. 7].

[0087] Example 3. Manufacturing process in industrial conditions

[0088] The method implemented in accordance with the invention has been validated in accordance with GMP requirements. Key process parameters and their limits or ranges are defined (and shown in Table 7). This made it possible to standardize the product manufacturing process for clinical applications. Reference values have been established for in-process quality control for the steps of the manufacturing process. The used culture method allows obtaining AT-MSCs with comparable biological properties. The validation of the manufacturing process was carried out in the manufacturing site - the ATMP Manufacturing Facility operating in the GMP standard. The quality control values obtained for 3 batches during the manufacture of MesoCellA product in a GMP certified ATMP Manufacturing Facility are shown in the table below.

[0089] Table 7. Quality control of the MesoCellA manufacturing process comprising AT-MSCs as active substance.

[0090]

[0091] AT-MSCs immediately after isolation (samples: 20210318 / 1 A, 20210322 / 1 / 2A, 20210322 / 2 / 3A) as well as after long-term culture (samples: 20210318 / 1 B, 20210322 / 1 / 2B, 20210322 / 2 / 3B) in accordance with the manufacturing process shown in Fig. 1 , maintain the correct (normal, unchanged) karyotype. It was confirmed that short- and long-term cultured AT- MSCs do not contain any numerical or structural changes within the chromosomes, which confirms the genetic stability of AT-MSCs at the beginning and at the end of the culture process and provides an argument for the safety of AT-MSCs (being the active substance of MesoCellA) for further clinical use. Analysis was performed using an Agilent ISCA 8x60K V2 oligonucleotide matrix. The results of the karyotype test are presented in Table 8.

[0092] Table 8. Genetic analysis of AT-MSCs.

Claims

Claims1. A method for preparing autologous mesenchymal / stromal stem cells from adipose tissue (AT-MSCs), characterized in that: a) an adipose tissue collected as a lipoaspirate is suspended in a solution isotonic to human cells, preferably in a buffered saline (PBS) comprising a mixture of antibiotics, preferably including antibiotics and an antifungal agent, and then the aqueous fraction and the lipid fraction are separated, and further the lipid fraction is resuspended in a fresh isotonic solution, preferably a PBS salt comprising a mixture of antibiotics, preferably including antibiotics and antifungal agent, wherein the washing is repeated several times, especially 3 to 6 times, preferably until the red color disappears, b) the adipose tissue sample obtained in step a) is digested with a collagenase having an enzymatic activity of 0.1 to 0.5 PZ U / ml, preferably 0.2 PZ U / ml of the reaction mixture, wherein the digestion reaction is carried out at 30-40°C for 10-60 minutes, preferably at 37±1°C for 35 to 40 minutes, and then SVF cells are separated, preferably by centrifugation, and further washed with isotonic solution and SVF cells are again separated, c) SVF cells are cultured in a first human cell culture medium comprising 1-20% human platelet lysate (hPL), preferably 5-10% hPL, and preferably additionally a mixture of antibiotics and antifungal agent, wherein the preferred concentration of antibiotics and antimycotics is 1% (100 U / ml penicillin, 100 pg / ml streptomycin, 0.25 pg / mL amphotericin B); wherein the culture is carried out at 35-40°C, preferably 37°C in an atmosphere with CO2 enriched to 5% and O2 in the range of 0.5-23%, preferably 21%, and over a period of 12-72 hours, preferably 24-48 hours, after which non-adherent cells are rinsed off, fresh culture medium is added and the culture is continued for 3-6 ± 2 days, preferably 4-5 ± 2 days counted from the moment of culture establishment, d) the cells obtained in step c) are cultured in a culture medium comprising 1-20% human platelet lysate (hPL), preferably 5-10% hPL, and preferably without the addition of a mixture of antibiotics and antifungal agent, wherein the culture is carried out at 35-40°C, preferably 37°C, under air with CO2 content enriched to 5% and O2 in the range of 0.5-23%, preferably 21 % and over a period of 6-20 ± 2 days, preferably 8-17 ± 2 days, counted from the moment of culture establishment, after which AT-MSCs cells are recovered from the surface of the cultured vessel by means of a passage, e) the cells obtained in step d) are washed with an isotonic solution, preferably a PBS salt solution, and then separated, preferably by centrifugation, and suspended in a solution of a carrier comprising a solution isotonic to human cells, preferably saline (with 0.9% NaCI) andHSA in a concentration in the range of 1% to 20%, preferably a solution with 10% HSA concentration obtained by mixing 0.9% NaCI solution with 20% HSA solution in a 1 :1 ratio.

2. The method according to claim 1 , characterized in that in step a) the lipoaspirate is mixed with the solution isotonic to human cells, preferably PBS salt, in a volume ratio from 1 :1 to 1 :10.

3. The method according to claim 1 , characterized in that, in step b), collagenase is used, preferably of GMP or cGMP grade, constituting a mixture of class I and II collagenase, wherein, preferably during the digestion, collagenase activity is inhibited by cooling of test tubes with digested adipose tissue (during centrifugation at 4-25 ± 1 °C, preferably 17 ± 1 °C).

4. The method according to claim 1 , characterized in that, in step b), SVF cells are suspended in the solution isotonic to human cells, preferably saline, and then separated by centrifugation, wherein washing with the isotonic solution is repeated at least twice.

5. The method according to claim 1 , characterized in that, in step c), the culture is carried out until the first passage.

6. The method according to claim 1 characterized in that, in step d), the culture is repeated for consecutive 2 to 7 passages, preferably 2 to 4 passages.

7. The method according to claim 1 characterized in that the product is prepared from AT- MSCs cells at passage no earlier than 2 and no later than 7, preferably no earlier than 3 and no later than 5.

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