A method for stimulating cell growth and proliferation of cells of interest, and a method for preparing liver or pancreas grafts.

Abstract

The present invention provides, inter alia, a method for preparing a liver or pancreas graft, comprising the following steps: 2a) providing a carrier matrix suitable for tissue engineering and a cell suspension comprising viable hepatocytes and / or islet cells in a standard culture medium A; 2b) loading the cell suspension onto the carrier matrix; 2c) incubating the loaded matrix in either -standard culture medium A or -standard culture medium A and culture medium B comprising 0.5%-5% secretome at about 37°C for a first incubation time T1 of at least 10 hours; 2d) replacing the culture medium of step 2c) with a culture medium B comprising standard culture medium A and 0.5%-5% secretome; 2e) incubating the loaded matrix in culture medium B at about 37°C for a second incubation time T2 of at least 24 hours; 2f) washing the loaded matrix of step e) with standard culture medium A to remove at least a portion of the secretome.

Description

Technical Field

[0001] The present invention relates to a method for stimulating the cell growth and proliferation of cells of interest, specifically hepatocytes and / or pancreatic islet cells, according to claim 1, and a method for preparing a liver or pancreatic graft according to claim 2.

Background Art

[0002] Every day, people are dying from organ failure. Unfortunately, currently, patients suffering from conditions leading to organ failure have limited treatment options. Even if conventional drug therapies may help delay or alleviate the symptoms of the disease, they often cannot restore the full functionality of the damaged tissue. For these reasons, in the case of end-stage organ failure, transplantation of healthy organs or at least somewhat healthy organ tissue from suitable tissue donors is the ultimate treatment option for many patients.

[0003] For example, transplantation of healthy liver or pancreatic tissue has proven successful in many patients suffering from severe pancreatic or liver diseases. However, the shortage of suitable tissue donors remains a significant obstacle. In attempts to find alternative treatment methods, cell-based therapies are gaining importance. In these cell-based therapies, tissue from the patient is removed, processed into viable single cells, and cultured to obtain a large population of viable cells. The cells are then transplanted as such to facilitate the body's natural regeneration process and contribute to the regeneration of organ failure or damaged tissue.

[0004] In the field of treating liver diseases, hepatocyte transplantation is becoming increasingly important as an alternative or symptomatic treatment to orthotopic liver transplantation for congenital or acute liver diseases. However, the difficulties in culturing hepatocytes (which means collecting cells from the patient's own tissue for easy culturing and proliferation in the laboratory) and the limited long-term viability of transplanted cells represent significant obstacles to the wider application of this cell approach.

[0005] A recent and intriguing cell-based approach is the simultaneous transplantation of hepatocytes and pancreatic islets: It is known that hepatocyte proliferation and survival are stimulated by co-culture with islet cells (or "islet cells"). This concept is described, for example, in EP3010556B1. In particular, beta cells (insulin-producing cells) have been found to enhance the proliferation rate of hepatocytes by up to 300%. On the downside, this means that the production of autologous liver grafts (i.e., carrier matrices loaded with viable hepatocytes) requires not only the excision of liver tissue from the patient, but also the separate excision of the patient's pancreatic tissue by surgical or laparoscopic means. The excised tissue is then processed outside the patient's body to obtain both hepatocytes and islet cells for co-culture. If a sufficient number of hepatocytes and islet cells can be cultured in vitro, a second surgery is performed in which one or more suitable carriers loaded with the cultured cell lines are inserted subserosa or at another suitable location in the patient's small intestinal mesenteric ligament. However, all surgeries carry unavoidable risks, and pancreatic tissue resection in particular carries a significant risk of pancreatic duct damage, which can lead to acute inflammation (pancreatitis) or dangerous or life-threatening pancreatic fistula formation. Therefore, there is a need for a method that can stimulate hepatocyte growth—ideally without the need for co-culture with pancreatic cells.

[0006] Therefore, the object of the present invention is to provide a safer and less time- and cost-intensive method for culturing cells, particularly liver and / or pancreatic cells, taking into consideration their use in the field of regenerative medicine, particularly in the field of treating chronic hepatitis. More specifically, the present invention aims to provide a safe and cost-effective method for preparing grafts, particularly liver or pancreatic grafts.

[0007] This objective is achieved by the methods of claims 1 and 2. Preferred embodiments are subject to the dependent claims.

[0008] The inventions described herein encompass methods for stimulating cell growth and proliferation by incubation of cells of interest in a cell growth medium or fluid together with a secretome derived from mesenchymal stem cells. Specifically, the cells of interest (or “target cells”) are hepatocytes and / or islet cells of Langerhans (including hepatocyte progenitor cells), i.e., pancreatic cells. With respect to both hepatocytes and islet cells of Langerhans, incubation together with a secretome has been found to be particularly effective in promoting proliferation and increasing the viability of these cells. Since hepatocytes are known to be very difficult to culture, the methods of the present invention are particularly suitable for stimulating cell growth and proliferation of hepatocytes.

[0009] In particular, in one embodiment, the present invention involves the following steps: 1a) Prepare the viable cells of interest; 1b) The cells of interest are as follows: - Standard culture medium A, or - Standard culture medium A and culture medium B containing 0.5% to 5% secretome. Incubate at approximately 37°C for a first incubation time T1 of at least 10 hours; 1c) Replace the culture medium from step 1b) with culture medium B containing standard culture medium A and at least 0.5% to 5% secretome; and 1d) Incubate the cells of interest in culture medium B for at least 24 hours for a second incubation period T2. This provides a method for stimulating the cell growth and proliferation of cells of interest, specifically hepatocytes and / or islet cells.

[0010] This invention is based on the inventors' discovery that not only the presence of mesenchymal stem cells (MSCs), but also the MSC secretome, positively influences cell proliferation in vitro and in vivo, even in sensitive cells such as hepatocytes. Within the scope of this application, the term "secretome" refers to factors released from mesenchymal stem cells (MSCs), particularly platelet-derived growth factor (PDGF), which is found in bone marrow, muscle, and adipose tissue, as well as in Wharton's jelly, dental pulp, peripheral blood, skin, lungs, chorionic villi, menstrual blood, and breast milk. The method of this invention, which involves culturing cells of interest in a culture medium containing 0.3% to 5% MSC secretome for at least 24 hours, has been found to significantly enhance cell proliferation and viability.

[0011] Surprisingly, it was also found that these cells promote proliferation and survival: it is well known that the function of isolated primary hepatocytes is difficult to maintain when cultured in vitro, as these cells are susceptible to dedifferentiation, which leads to loss of hepatocyte function. However, developing a sufficient number of functional hepatocytes is a prerequisite for treating liver diseases that result in end-stage liver failure and therefore require liver cell transplantation and artificial liver support systems.

[0012] Accordingly, the inventors of the method of the present invention described herein were very excited to find that the treatment of hepatocytes by the method of the present invention increased hepatocyte growth and proliferation rates to the same extent as, or even higher than, when hepatocytes were co-cultured with islet cells of Langerhans (as mentioned earlier, the latter is so far one of the most effective known methods of hepatocyte stimulation). Therefore, the method of the present invention provides a novel method for stimulating hepatocyte growth and proliferation without the need to co-culture them with islet cells of Langerhans. In practice, this means that the production of autologous liver grafts or autologous liver cell transplants is achieved by excision, culture, and re-implantation of autologous hepatocytes without requiring additional surgical means for the excision of islet cells (i.e., pancreatic cells) from the patient for co-culture with hepatocytes.

[0013] While these new findings are promising, several new challenges need to be overcome when MSC secretomes are used to stimulate cell growth in clinical settings: it has been found that some factors of the MSC secretome also exert inhibitory effects on the microenvironment, such as cellular apoptosis, inflammation, pathological remodeling, or scar formation, depending on concentration and release kinetics. Furthermore, the growth-promoting effects exhibited by the MSC secretome could not be replicated (or at least not to the same extent) by delivery of any of the cytokines / growth factors identified in isolation.

[0014] Therefore, when using an MSC secretome to stimulate cell growth in vivo, it is necessary to achieve an appropriate balance between the promoting and inhibiting factors produced by the MSCs, and to maintain the desired effect after in vivo transplantation. This invention finds a way to overcome these challenges and presents a method that enables the safe use of an MSC secretome in the preparation of tissue grafts for later use in regenerative medicine.

[0015] Specifically, in a second embodiment, the present invention relates to a method for preparing a graft for use in regenerative medicine, comprising the following steps: 2a) Prepare a carrier matrix suitable for tissue engineering and a cell suspension containing viable hepatocytes in standard culture medium A; 2b) Load the cell suspension into the matrix; 2c) Incubate the loaded matrix in either standard culture medium A or culture medium B containing standard culture medium A and at least 0.5% secretome at approximately 37°C for an incubation time T1 of at least 10 hours; 2d) Replace the culture medium from step 2c) with culture medium B containing standard culture medium A and at least 0.5% secretome; 2e) Incubate the matrix in culture medium B at approximately 37°C for an incubation time T2 of at least 24 hours; 2f) Wash the matrix loaded with hepatocytes from step e) with standard culture medium A to remove at least a portion of the secretome. This provides a method that includes this.

[0016] The method according to the present invention involves the use of "Standard Culture Medium A". Cell incubation is carried out in any suitable cell growth medium using culture conditions that support the growth, differentiation, and protein synthesis of the cells of interest. Many commercially available culture media, such as Dulbecco's Modified Eagle Medium (DMEM), Williams E, RPMI 1640, Fischer, Iscove, and McCoy's, may be suitable for use as Standard Culture Medium A. This medium may be supplemented with additional salts, carbon sources, amino acids, serum and serum components, vitamins, minerals, reducing agents, buffers, lipids, nucleosides, antibiotics, adherents, and growth factors. Formulations for various types of culture media are described in various references available to those skilled in the art (e.g., Culture of Animal Cells, A Manual of Basic Techniques, 4th Ed., Wiley-Liss (2000)). The standard culture medium used in any of the culture steps of the present invention may or may not contain serum, whether under aerobic (normal oxygen) or hypoxic conditions. A preferred standard culture medium is Williams E medium, which is widely used in cell culture laboratories and is well known to those skilled in the art.

[0017] As used herein, the term “matrix” refers to a three-dimensional scaffold. A three-dimensional support or scaffold used for culturing cells may be of any material and / or shape that: (a) allows cells to adhere to it (or may be modified to allow cells to adhere to it); and (b) allows cells to grow in two or more layers (i.e., form a three-dimensional tissue). In this sense, the matrix serves as a three-dimensional template to which cells or tissue can be attached. This attachment may occur in vitro or in vivo. Furthermore, in relation to transplantation, the matrix also serves to position the graft and also as a placeholder for the tissue that is gradually formed in vivo. The matrix preferably has a sponge-like structure with pores of varying sizes, at least in part of which are connected to one another. In addition, the matrix does not necessarily need to be provided in an immutable shape. It may consist of multiple small patches or even have a paste-like structure.

[0018] Regardless of the use of the matrix as a carrier, an important aspect of the present invention is the culture of cells of interest—particularly hepatocytes and / or islet cells—in culture medium B containing a secretome. In particular, it is preferable that culture medium B contains a secretome derived from umbilical cord mesenchymal stem cells (hucMSCs). Similar to MSCs from bone marrow, human umbilical cord-MSCs (hucMSCs) have high regenerative capacity and low immunogenicity, and hucMSCs can be obtained by non-invasive procedures and are easily cultured, which makes them potentially superior to MSCs from other sources.

[0019] The MSC secretome is provided as either MSC-conditioned medium (CM) or purified MSC-derived extracellular vesicles (EVs). Surprisingly, particularly high proliferation and survival rates were seen when cells, especially hepatocytes, were seeded in conditioned medium from MSCs cultured under hypoxic conditions instead of using conditioned medium from the same cells cultured under normoxic conditions. As used herein, hypoxic conditions are characterized by a lower oxygen concentration when compared to the oxygen concentration of ambient air (ambient air contains approximately 15% - 20% oxygen). Preferably, the hypoxic conditions are present at 3% - 5% oxygen. The hypoxic conditions can be created and maintained by using a culture apparatus that allows for control of the ambient gas concentration, such as an anaerobic chamber.

[0020] Without wishing to be bound by theory, hypoxic exposure is thought to enhance their therapeutic efficacy by activating several signaling pathways, including hypoxia-inducible factor (HIF), a major transcription factor that regulates the expression of hundreds of genes to promote self-renewal of undifferentiated mesenchymal stem cells and to promote cellular adaptation to hypoxic conditions.

[0021] Thus, with respect to the method of the present invention, the secretome preferably is derived from mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells, cultured under hypoxic conditions.

[0022] Preferably, a commercially available secretome from human Wharton's jelly stem cells is used. This secretome has been found to be particularly effective in stimulating hepatocytes.

[0023] With respect to the concentration of the secretome in culture medium B, optimal growth stimulation of normal cells, especially hepatocytes, was found when using secretome from MSCs, preferably human Wharton's jelly stem cells, at a concentration of 0.5% - 5%, preferably 0.5% - 3%, more preferably approximately 1%. For at least hepatocytes, a concentration of secretome in culture medium B higher than 5% was not beneficial and actually decreased survival rates.

[0024] With respect to both methods of the present invention - that is, with respect to providing cell growth and proliferation of cells of general interest, particularly hepatocytes and / or pancreatic islet cells - and with respect to providing grafts for use in regenerative medicine, it is preferred that the first incubation time T1 is 17 to 27 hours, preferably about 24 hours.

[0025] The second incubation time T2 is usually longer than the first incubation time T1 and is preferably within the range of 35 to 50 hours, preferably about 48 hours.

[0026] These incubation times have been found to be most effective for obtaining a large cell population and - when preparing grafts - for achieving a high cell attachment rate to the matrix. In particular, with respect to hepatocyte growth, since delicate hepatocytes usually require more growth and stimulation time, the first incubation time is about 24 hours and the second incubation time is about 48 hours.

[0027] It is generally preferred to load the matrix with cells of interest before incubating the cells in the secretome-containing culture medium B, although culturing the cells of interest in the presence of the MSC secretome can also occur before the matrix as a carrier is implanted. Considering the use of the matrix loaded with cells as a graft, the method of the present invention requires that both the matrix and the cells loaded therein are subjected to a washing process to remove the secretome. Next, the resulting graft can be re-implanted into the body to take over the function of the damaged organ in the patient.

[0028] Animal studies have confirmed that cells cultured in the presence of MSC secretomes and processed inline by the present invention maintained high cell viability and complete homing to their target sites after in vivo transplantation. Since the secretomes are removed from the graft at least partially before transplantation, there is no concern whatsoever regarding the potential negative or inhibitory effects of secretomes in the patient's body. Aside from potential immune rejection and pathogen transmission, the removal of secretomes from the cell-loaded matrix when non-allogeneic secretomes are used also overcomes regulatory issues associated with the transplantation of stem cells or stem cell secretomes.

[0029] Therefore, when applied, for example, to the production of liver grafts, the method of the present invention enables a safer and less cost-intensive method for producing grafts using a large viable population of hepatocytes or hepatocyte-like cells compared to standard methods involving the excision and co-culture of hepatocytes and islet cells of Langerhans.

[0030] In a preferred embodiment, in step 2c) of the graft preparation method of the present invention, the loaded matrix is ​​first incubated in standard culture medium A for about 30 minutes, and then the matrix is ​​incubated in culture medium B for an incubation time T1. This has been shown to further enhance cell adhesion. This pretreatment step is also preferred for the method according to claim 1 of this application.

[0031] Regarding the washing step (2g), washing is preferably carried out by a first culture medium exchange step in which culture medium B is replaced with fresh standard culture medium A. This first culture medium exchange step is preferably followed by at least one second culture medium exchange step in which previously added standard culture medium A is replaced with fresh standard culture medium A.

[0032] In a particularly preferred embodiment, the washing step 2g) is carried out as described in the preceding paragraph, and more specifically, the first and / or second culture medium exchange step is: i) Transfer the matrix into a new container containing fresh standard culture medium A; and ii) Repeat step i) at least once, preferably at least twice, most preferably three times. It will be implemented by [company name].

[0033] Instead of placing the matrix into a new container with fresh standard culture medium A, the matrix is ​​also kept in the original container while the culture medium is carefully aspirated, for example, using a pipette, and replaced with fresh medium carefully added around the matrix.

[0034] This preferred method of washing the cell-loaded matrix after secretome treatment has proven to be particularly gentle, which makes it very suitable for sensitive cells such as hepatocytes.

[0035] To enable not only proper cell growth but also secretome removal, the matrix preferably includes a porous scaffold, preferably a sponge-like structure having pores that are at least partially interconnected. A porous scaffold refers to a mesh structure with pores through which molecules diffuse in and out. The porous nature of the matrix facilitates cell adhesion and the flow of nutrients through the matrix.

[0036] With respect to the composition, the matrix may be selected depending on the tissue to be treated, the type of injury to be treated, the desired treatment duration, the lifespan of the cell culture in vivo, and the time required to prepare the matrix. The matrix may comprise a variety of polymers, whether natural or synthetic, charged (i.e., anionic or cationic) or uncharged, biodegradable or non-biodegradable.

[0037] The term "biodegradable" refers to a material that can be converted into metabolizable products in a living organism (or bodily fluids or cell cultures derived from a living organism). Biologically degradable polymers include, for example, bioabsorbable and / or bioerosive polymers. "Bioerosive" refers to the ability to be soluble or suspended in biological fluids. "Bioabsorbable" means the ability to be taken up by the cells, tissues, or fluids of a living organism.

[0038] Examples of biodegradable polymers include natural polymers such as fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginic acid, or polyamino acids such as poly-lysine. Alternatively, the matrix may be made from synthetic polymers, non-limiting examples of which include polylactide (PLA), polyglycolide (PGA), poly(lactide-coglycolide) (PLGA), poly(caprolactone), polydioxanone, trimethylene carbonate, polyhydroxyalconate (e.g., poly(hydroxybutyric acid), poly(ethylglutamic acid), poly(GTH iminocarbonylbisphenol A iminocarbonate), poly(orthoester), and polycyanoacrylate).

[0039] In a preferred embodiment, the matrix consists of one or more polymers selected from the group consisting of PLA, PGA, a mixture thereof (PLGA), and natural polymers such as collagen. A suitable basic polymer material for the matrix is ​​a 50:50 mixture of PLA and PGA—that is, a polymer mixture having a content of about 50 mol% lactic acid (PLA) and about 50 mol% glycolic acid (PGA). Such a 50:50 mixture can be purchased, for example, from Evonik Industries AG (Essen, Germany) under the trade name RESOMER® RG502, or from Durect (Cupertino, CA, USA) under the trade name Lactel B6007-1.

[0040] However, preferably, the matrix comprises a basic polymer structure consisting mainly or entirely of PLA, particularly PLLA (e.g., Lactel B6002-2). “Mainly” means that preferably the basic polymer structure consists of at least 85%, preferably at least 90%, of PLA. The basic polymer structure is preferably coated with a natural polymer, particularly collagen.

[0041] The preparation methods for preparing the matrix from the synthetic polymers mentioned earlier are well known in the art. One possibility is the use of salt leaching techniques, as described, for example, in EP2256155.

[0042] It is more preferable that the matrix contains or is at least partially coated with at least one natural polymer selected from the group consisting of collagen, gelatin, laminin, fibrinogen, albumin, chitin, chitosan, agarose, hyaluronic acid, arginate, and mixtures thereof, and therein, collagen is preferred. The natural polymer provides additional stability, hydrophilicity to the matrix, and facilitates cell adhesion and proliferation. A matrix with a collagen coating can be obtained, for example, by covering the matrix with a 1% collagen solution or by immersing the matrix therein and subsequently freeze-drying it.

[0043] In some embodiments, the matrix may include a hydrogel, which is typically a hydrophilic polymer network filled with water. Hydrogels have the advantage of selectively triggering polymer swelling. Depending on the composition of the polymer network, swelling of the matrix can be triggered by a variety of stimuli, including pH, ionic strength, heat, electricity, ultrasound, and enzymatic activity. Non-limiting examples of polymers useful in hydrogel compositions include, in particular, poly(lactide-co-glycolide); poly(N-isopropylamide); poly(methacrylate-g-polyethylene glycol); polymers formed from polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol, as well as natural compounds such as chondroitan sulfate, chitosan, gelatin, and fibrinogen, or mixtures of synthetic and natural polymers, e.g., chitosan-poly(ethylene oxide). These polymers can be reversibly or irreversibly crosslinked to form gels adaptable to forming the matrix.

[0044] To facilitate cell adhesion and growth throughout the matrix, the latter is preferably hydrophilic, at least on its surface. As used in connection with this invention, the term "hydrophilic" means that the water contact angle of the surface area on the matrix is ​​less than 75°. A hydrophilic surface is obtained, for example, by increasing the content of hydrophilic polymers such as gelatin or collagen in the matrix material, or by coating the matrix with them, and / or by post-treatment methods such as plasma treatment. The term "plasma" generally refers to an excited and radicalized gas, i.e., a conductive process gas accompanied by electrons and ions. Plasma is generally produced by electrodes in a vacuum chamber (the so-called "RF plasma method"), but can also be produced by capacitive or inductive methods, or by microwave radiation. Further details in this regard can be found, for example, in WO2017 / 032837, the contents of which are incorporated herein by reference.

[0045] Considering its subsequent use as a graft, it is preferable that the matrix be sterilized before the cells of interest are seeded. For this purpose, it is preferable to expose the matrix to a hydrogen peroxide-containing environment at a temperature of 40°C to 50°C or lower.

[0046] A hydrogen peroxide-containing environment can be provided either by H2O2 plasma treatment or by placing the matrix to be sterilized in a vacuum chamber together with a hydrogen peroxide source, preferably a 30% H2O2 solution. The application of vacuum within the vacuum chamber causes the hydrogen peroxide to sublimate, creating a hydrogen peroxide-containing atmosphere. This sterilization technique, developed by the inventors of this application, enables sterilization of heat and / or UV-sensitive tissues, particularly polymer scaffolds, without impairing their hydrophilic properties or damaging their polymer structure. Therefore, it is particularly well-suited for sterilizing matrices for use in the method of the present invention.

[0047] The processing time for the matrix in an H2O2-containing atmosphere is at least 1 minute, preferably at least 5 minutes, more preferably 5 to 20 minutes, at a temperature of 40°C to 50°C. Alternatively, if sterilization is performed at a temperature of 40°C or lower, the processing time is preferably at least 4 hours, more preferably about 8 to 12 hours, and most preferably about 10 hours.

[0048] In addition, the negative pressure applied in the vacuum chamber is preferably in the range of 6 to 12 mbar, more preferably 8 to 10 mbar, and most preferably about 9 mbar.

[0049] To increase hydrophilicity and thereby improve cell adhesion, the matrix is ​​plasma-treated before cells are seeded. Plasma treatment is preferably carried out by exposing the matrix to a reactive gas plasma at a temperature below 50°C for at least 1 minute, preferably at least 5 minutes, and more preferably about 10 minutes. For plasma treatment, the pressure is preferably 10 -2 ~10 -6It is within the range of bar, more preferably within the range of 0.1 to 1.0 mbar.

[0050] The matrix is ​​preferably provided in a sheet-like form, i.e., having a thickness thinner than the length and width of the matrix. Preferably, the matrix is ​​also elastically deformable so that it can be folded or rolled up. Thus, the matrix, with cells attached to it, can be introduced into the body by a laparoscopic tool, such as a trocar. The external shape of the matrix (when viewed in a top view or longitudinal section) can be of any kind, e.g., rectangular, square, circular, elliptical, etc., and can also be cut into any desired shape. Preferably, the external shape of the transverse section is circular or elliptical to avoid any sharp corners.

[0051] Alternatively, the matrix may also be provided in the form of a hydrogel or consist of several small patches. Such patches may be very small so that the cell-loaded matrix can be transplanted or applied in the form of a paste-like structure.

[0052] Considering the use of a matrix in which cells are seeded as a graft, the matrix should contain 1 cm 3 Preferably, 250,000 cells, and more preferably at least 500,000 cells, are loaded each time.

[0053] For use as a graft, the matrix should contain at least 1 × 10⁶ 6 It is preferable that a certain number of cells are loaded. For a liver graft, for example, it is preferable that the matrix be loaded with at least 1 million hepatocytes.

[0054] In a further embodiment, the present invention also provides a method for treating a patient requiring a liver transplant, comprising the following steps: i. Take liver tissue from the patient by invasive or laparoscopic surgical means; ii. The liver tissue is processed in the laboratory to select hepatocytes; iii. Seed the hepatocytes onto a matrix, preferably a matrix consisting of polyglycolic acid (PGA), polylactic acid (PLA), a mixture thereof (PLGA), and collagen; iv. Stimulate the hepatocytes by incubating them for at least 24 hours in a culture medium containing at least 0.5% MSC-derived secretome, either before or after seeding the hepatocytes in the matrix; v. Wash the matrix with attached hepatocytes using standard culture medium to remove at least some of the secretome; vi. The washed hepatocytes are loaded into a matrix graft, which is then transplanted into the patient's body, preferably into the mesentery of the small intestine. Regarding methods including

[0055] Before loading the hepatocytes, the matrix is ​​preferably plasma-treated and / or sterilized with the help of H2O2 as described in the previous section. [Examples]

[0056] material Stem cells and island cells It was newly isolated from cirrhotic liver / pancreatic tissue of rats or human patients. Secretome Human umbilical cord culture medium (CM) was obtained from the Stem Cell and Cancer Institute, PT. Kalbe Farma, Tbk., Jakarta, Indonesia, under normal and hypoxic conditions.

[0057] method Preparation of standard culture medium A: HEPES was added to Williams E medium in an amount that yielded a final concentration of 50 mM. Next, L-glutamine and sodium pyruvate were added, followed by fetal bovine serum (FBS) in an amount equivalent to 10% of the total volume. Finally, an antibiotic / antifungal substance was added in an amount equivalent to 1% of the total volume. The resulting culture medium was thoroughly mixed.

[0058] [Table 1]

[0059] Preparation of culture medium B containing 1% secretome 2 mL of secretome was added to 198 mL of standard culture medium A, which was prepared as described above, and thoroughly mixed.

[0060] Matrix preparation from PLLA PLLA matrices were prepared using a salt leaching technique with sodium chloride (NaCl) microparticles as a porosity agent. NaCl microparticles were sieved, and particles with a diameter range of 355 μm to 425 μm were used. PLLA (1 g) was dissolved in chloroform (5-5.3 mL), and the sieved NaCl microparticles (9.0 g) were placed in an aluminum pot. The PLLA solution was poured into the aluminum pot and mixed with the NaCl microparticles. The mixture was air-dried in a stream for 48 hours. Next, the aluminum pot was broken open, and the dried PLLA / NaCl block was peeled off. The PLLA / NaCl block was dried in a vacuum for another 48 hours, and the NaCl microparticles were dissolved by immersion of the dried PLLA / NaCl block in deionized water, with the deionized water being changed every hour. This immersion washing was continued until the weight of the PLLA sponge remained constant. The preparation of the PLLA matrix was complete when the PLLA sponge was completely dry.

[0061] Collagen matrix coating The matrix prepared from PLLA was cut to the desired size and then immersed in a 1% collagen solution (fetal bovine atelocollagen (KOKEN, Japan, IPC-50)) to fill all the pores of the PLLA matrix with the collagen solution to ensure a uniform coating.

[0062] After complete immersion, the matrix was removed from the collagen solution, placed in a centrifuge tube, and centrifuged at 1000g at 4°C for 5 minutes on each side (i.e., a total of 10 minutes) using a Low Speed ​​Refrigerated Centrifuge (Tomy, AX501) to remove all excess collagen solution. The collagen-coated matrix was then placed in a large freezer (New Brunswick, Innova U101) at -80°C for at least 4 hours, and subsequently freeze-dried under a vacuum of less than 5 Pa using a Vacuum Freeze Dryer (EYELA, FDU-2200) for at least 24 hours. The aforementioned final freeze-drying step preserved the hydrophilicity of the coated collagen layer.

[0063] The collagen-coated matrix was stored in a desiccator until use.

[0064] Plasma cleaning of the matrix As described above, the prepared collagen-coated matrix was further cleaned by plasma cleaning using oxygen-containing gas plasma. The matrix was placed in the vacuum chamber of a Plasma Machine (Company Diener), and the vacuum chamber was degassed to a pressure of 0.4 mbar before introducing the oxygen gas plasma into it. The processing time was 5 to 10 minutes. This plasma treatment not only cleans the matrix surface but also activates it, thereby increasing the hydrophilicity of the surface.

[0065] Sterilization of the matrix The plasma cleaning matrices were sterilized by placing them in an H2O2-containing environment for at least 10 minutes (at a temperature of 40°C to 50°C) or for up to 12 hours at a temperature below 40°C. The H2O2-containing environment was created in a vacuum chamber by placing the matrices in a Tyvek bag in a chamber along with an open flask containing a 30% H2O2 solution (Merck, Darmstadt, Germany, #108597), and then vacuuming the chamber to sublimate the H2O2. The processing time depends heavily on the pressure in the chamber and the concentration of the H2O2 solution. Preferred processing times are 5 to 20 minutes at temperatures around 40°C and 8 to 12 hours, preferably about 10 hours, at temperatures below 40°C.

[0066] Cell isolation Perfused liver tissue was cut into small pieces and passed through a 1mm mesh tea strainer using a spatula. The strainer was washed with C / H solution, and the collected cell suspension was passed through a 100-micron cell sieve, followed by centrifugation at 130G for 10 minutes. The supernatant was aspirated using a capillary pipette, and the pellet was resuspended in Williams E Medium (Williams E Med containing 10% FBS). The centrifugation and resuspension steps were repeated several times. The resulting liver cell suspension was incubated at 37°C and 5% CO2 until ready for further use. Cell count and viability were measured using trypan blue.

[0067] Cell mixture analysis was performed in 3D format (on a collagen-coated PLLA matrix). The same procedure was applied to pancreatic tissue to obtain a cell suspension of islet Langerhans cells.

[0068] Sample preparation Five sample groups were prepared. Each group incorporated three PLLA-matrices coated with 1% collagen. The matrices were plasma-treated and sterilized according to the method described above, and then cell suspensions in standard culture medium A (hepatocytes, or 1 × 10⁶ cells) were placed on each matrix.6 Cells were seeded by adding 600-700 μL of either a mixture of hepatocytes and islet cells in a ratio of 50,000 hepatocytes to 50,000 islet cells (cell density: 1,000,000 cells / matrix).

[0069] Group 1: Hepatocytes and secretome Group 2: Secretomes derived from hepatocytes and MSCs cultured under hypoxic conditions Group 3: Hepatocytes, islet cells, and secretomes Group 4: Hepatocytes only (Control 1) Group 5: Hepatocytes and islet cells (control 2)

[0070] Sample processing For control groups 4 and 5, a matrix seeded with hepatocytes (group 4) or hepatocytes and islet cells (group 5) was incubated in standard culture medium A (without the addition of culture medium B) that did not contain secretome.

[0071] For groups 1, 2, and 3: The matrix with the seeded cells was incubated at 37°C and 5% CO2 for 30 minutes. Next, standard culture medium A surrounding the matrix was aspirated using a micropipette, and 600 μL of culture medium B containing 1% secretome was added to the matrix under sterile conditions. The cells in the aspirated medium were counted using a hemocytometer (manual counting).

[0072] On the second day (24 hours later), under sterile conditions, all remaining culture medium was aspirated under sterile conditions, and 1 mL of fresh culture medium B was added. The cells in the aspirated culture medium were counted again using a hemocytometer (manual counting).

[0073] Procedure for rinsing off secretome The following washing procedure was performed on all matrices, including the control group, on day 4 (63-87 hours after sowing): The matrix was transferred to a new well, and 1 mL of fresh standard culture medium A was added dropwise around the matrix using a micropipette to wash away the secretome. This procedure was repeated three times.

[0074] After transferring the matrix to a new well, the number of cells remaining in the previous well was counted manually. The adhesion ratio was determined using the following formula: The number of cells on the matrix minus the number of cells remaining in the well equals the number of adherent cells on the matrix.

[0075] result While further research is still ongoing, the in vitro trial mentioned earlier yielded the following results: [Table 2]

[0076] The numbers above indicate the following order regarding adhesion rates: Group 3 > Group 2 > Group 1 ≒ Group 5 > Group 4

[0077] For group 3, i.e., a matrix seeded with hepatocytes and islet cells and treated with 1% secretome, the highest adhesion rate was obtained. Groups 2 (hepatocytes and secretome induced under hypoxic conditions), 1 (hepatocytes and secretome), and 5 (control group using a matrix seeded with hepatocytes and islet cells) showed similar adhesion rates, but were still significantly higher than the matrix of control group 4, which was seeded with hepatocytes only.

[0078] In vitro experiments demonstrated that MSC secretomes significantly enhance the viability and adhesion of hepatocytes on the matrix. Culture of hepatocytes in a culture medium containing MSC secretomes resulted in better adhesion and proliferation compared to co-culture of hepatocytes and islet cells. The best stimulation was achieved by combining co-culture with islet cells in a culture medium containing MSC secretomes.

[0079] The addition of secretomes derived from MSCs under both normal and hypoxic conditions enhanced adhesion rates, but secretomes derived from MSCs under hypoxic conditions were more beneficial.

[0080] A key finding was that a matrix seeded with hepatocytes and treated with secretome derived from MSCs cultured under normal conditions showed adhesion rates similar to—or even slightly better than—a matrix seeded with a mixture of hepatocytes and islet cells (without secretome addition). A matrix seeded with hepatocytes and treated with secretome derived from MSCs cultured under hypoxic conditions even showed clearly superior adhesion rates. This suggests that the addition of secretome, particularly secretome obtained under hypoxic conditions, is a valid alternative to co-seeding with islet cells to stimulate cell growth and hepatocyte adhesion. This alternative has the advantage of not requiring the harvesting of pancreatic tissue for liver graft production. Some aspects of the present invention are described below. 1. Follow these steps: 1a) Prepare the viable cells of interest; 1b) The cells of interest are as follows: - Standard culture medium A, or - Standard culture medium A and culture medium B containing 0.5% to 5% secretome. Incubate at approximately 37°C for a first incubation time T1 of at least 10 hours; 1c) Replace the culture medium from step 1b) with culture medium B containing standard culture medium A and 0.5% to 5% secretome; and 1d) Incubate the cells of interest in culture medium B for at least 24 hours for a second incubation period T2. A method for stimulating the cell growth and proliferation of cells of interest, specifically hepatocytes and / or islet cells of Langerhans, by performing the following actions. 2. Follow these steps: 2a) Prepare a carrier matrix suitable for tissue engineering and a cell suspension containing viable hepatocytes and / or islet cells in standard culture medium A; 2b) Load the cell suspension onto the carrier matrix; 2c) Load the matrix, - Standard culture medium A, or - Standard culture medium A and culture medium B containing 0.5% to 5% secretome. Incubate at approximately 37°C for a first incubation time T1 of at least 10 hours; 2d) Replace the culture medium from step 2c) with culture medium B containing standard culture medium A and 0.5% to 5% secretome; 2e) Incubate the loaded matrix in culture medium B at approximately 37°C for a second incubation period T2 of at least 24 hours; 2f) Wash the loaded matrix from step e) with standard culture medium A to remove at least a portion of the secretome. A method for preparing a liver or pancreas graft, which includes [a specific substance]. 3. The method according to item 1 or 2, wherein the culture medium B contains a secretome derived from umbilical cord mesenchymal stem cells. 4. The method according to item 3, wherein the secretome is derived from umbilical cord mesenchymal stem cells cultured under hypoxic conditions. 5. The method according to any one of items 1 to 4, wherein the first incubation time T1 is 17 to 27 hours, preferably about 24 hours. 6. The method according to any one of items 1 to 5, wherein the second incubation time T2 is 35 to 50 hours, preferably about 48 hours. 7. The method according to any one of items 1 to 6, wherein the culture medium B contains secretome in an amount of 0.5% to 3%, preferably about 1%. 8. The method according to item 2, wherein in step 2c), the loaded matrix is ​​first incubated in standard culture medium A for about 30 minutes, and then the loaded matrix is ​​incubated in culture medium B for a first incubation time T1. 9. The method according to item 2 or 8, wherein the washing step 2f) comprises a first culture medium exchange step in which culture medium B is replaced with fresh standard culture medium A, and preferably a second culture medium exchange step in which at least the previously added standard culture medium A is replaced with fresh standard culture medium A. 10. The first and / or second culture medium exchange step is: i) Transfer the matrix into a new container containing fresh standard culture medium A; and ii) Repeat step i) at least once, preferably at least twice, most preferably three times. The method described in item 9, as implemented by [company name]. 11. The matrix is ​​prepared before the cells are seeded in step 2b) as follows: At a temperature of -40°C to 50°C, for at least 1 minute, preferably at least 5 minutes, more preferably 5 to 20 minutes, or At a temperature below -40°C, for at least 4 hours, more preferably 8 to 12 hours, and most preferably about 10 hours. The method according to item 2 or any of items 8-10, wherein the matrix is ​​sterilized by exposing it to H2O2 plasma or an H2O2-containing atmosphere over any of the above. 12. The method according to item 2 or any of items 8 to 11, wherein the matrix is ​​plasma-treated by exposing it to a reactive gas plasma at a temperature below 50°C for at least 1 minute, preferably at least 5 minutes, and more preferably about 10 minutes, before the cells are seeded in step 2b). 13. In the matrix, 1 cm 3The method according to item 2 or any of items 8-12, wherein at least 250,000 cells, preferably at least 500,000 cells, are loaded each time. 14. The matrix contains at least 1 × 10 6 The method described in item 2 or any of items 8-13, wherein individual cells are loaded. 15. The method according to item 2 or any of items 8 to 14, wherein the matrix comprises a basic polymer structure mainly or entirely composed of polylactic acid, which is preferably coated with collagen, by coating the basic polymer structure with a 1% collagen solution and subsequently freeze-drying it.

Claims

1. The following steps: 2a) Prepare a carrier matrix suitable for tissue engineering and a cell suspension containing viable hepatocytes and / or islet cells in standard culture medium A; 2b) Load the cell suspension onto the carrier matrix; 2c) Load the matrix, - Standard culture medium A, or - Standard culture medium A and culture medium B containing 0.5% to 5% secretome derived from mesenchymal stem cells. Incubate at approximately 37°C for a first incubation time T1 of at least 10 hours; 2d) Replace the culture medium from step 2c) with standard culture medium A and culture medium B containing 0.5% to 5% secretome derived from mesenchymal stem cells; 2e) Incubate the loaded matrix in culture medium B at approximately 37°C for a second incubation period T2 of at least 24 hours; 2f) Wash the loaded matrix from step 2e) with standard culture medium A to remove at least a portion of the secretome. A method for preparing a liver or pancreas graft, which includes [a specific substance].

2. The method according to claim 1, wherein the culture medium B contains a secretome derived from umbilical cord mesenchymal stem cells.

3. The method according to claim 2, wherein the secretome is derived from umbilical cord mesenchymal stem cells cultured under hypoxic conditions.

4. The method according to any one of claims 1 to 3, wherein the first incubation time T1 is 17 to 27 hours.

5. The method according to any one of claims 1 to 4, wherein the second incubation time T2 is 35 to 50 hours.

6. The method according to any one of claims 1 to 5, wherein the culture medium B contains secretome in an amount of 0.5% to 3%.

7. The method according to claim 1, wherein in step 2c), the loaded matrix is ​​first incubated in standard culture medium A for about 30 minutes, and then the loaded matrix is ​​incubated in culture medium B for a first incubation time T1.

8. The method according to claim 1 or 7, wherein the washing step 2f) comprises a first culture medium exchange step in which culture medium B is replaced with fresh standard culture medium A.

9. The first and / or second culture medium exchange step is: i) Transfer the matrix into a new container containing fresh standard culture medium A; and ii) Repeat step i) at least once. The method according to claim 8, as implemented by [the specified method].

10. The matrix is ​​prepared before the cells are seeded in step 2b) as follows: At a temperature of 40°C to 50°C for at least 1 minute, or At a temperature below 40°C for at least 4 hours, H ２ O ２ Plasma or H ２ O ２ The method according to any one of claims 1 or 7 to 9, wherein the matrix is ​​sterilized by exposing it to the contained atmosphere.

11. The method according to any one of claims 1 or 7 to 10, wherein the matrix is ​​plasma-treated by exposing it to a reactive gas plasma at a temperature of less than 50°C for at least 1 minute before the cells are seeded in step 2b).

12. In the aforementioned matrix, 1 cm ３ The method according to any one of claims 1 or 7 to 11, wherein at least 250,000 cells are loaded each time.

13. The matrix contains at least 1 × 10 ６ The method according to any one of claims 1 or 7 to 12, wherein individual cells are loaded.

14. The method according to any one of claims 1 or 7 to 13, wherein the matrix comprises a basic polymer structure consisting mainly or entirely of polylactic acid.

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