Cryopreservation system for functional parenchymal hepatic cells and cryopreservation elution method thereof

Abstract

The invention discloses a cryopreservation system for functional parenchymal hepatic cells and a cryopreservation elution method of the cryopreservation system, and belongs to the technical field of biological medicine, and the cryopreservation system comprises an equilibrium solution, a cryopreservation solution and an elution solution. The cryopreservation liquid comprises a cryoprotectant composition, an amino acid stabilizer and a buffer solution, and the cryoprotectant composition is at least two of dimethyl sulfoxide, propylene glycol, glycerol, polyvinylpyrrolidone and hydroxyethyl starch. The cryopreservation and elution method comprises the steps of balancing, cryopreservation and elution. According to the invention, by reducing the content of dimethyl sulfoxide or completely replacing dimethyl sulfoxide, the potential toxic damage to cells caused by higher content in the traditional cryopreservation liquid is reduced, and the cell survival rate is improved. And moreover, the use of animal-derived components is completely eliminated, the permeation injury and toxic injury possibly suffered by the cells in the cryopreservation and recovery processes are reduced, and the method has important significance in promoting the research and application of the functional parenchymal hepatic cells in the fields of drug screening, disease model construction, cell therapy and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a freezing system for functional hepatocytes and a freezing elution method thereof. Background Art

[0002] The technology of inducing the differentiation of functional hepatocytes in vitro has made significant progress in recent years. These technologies mainly focus on converting pluripotent stem cells (such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC)) or adult stem cells (such as mesenchymal stem cells (MSC)) into functional hepatocytes. There are many methods to induce the differentiation of hepatocytes in vitro, mainly including directed differentiation pathways, three-dimensional culture pathways, adult stem cell differentiation pathways and chemical small molecule induction pathways. These methods induce the differentiation of stem cells into functional hepatocytes by precisely regulating growth factors and small molecule compounds to meet the needs of drug screening, disease model construction and cell therapy.

[0003] Cryopreservation technology plays a key bridging role between the large-scale preparation and production of functional hepatocytes induced for differentiation in vitro and their subsequent applications. It not only ensures the quality and consistency of cells, improves production efficiency and transportation flexibility, but also supports large-scale application needs. Through cryopreservation technology, production and application can be better connected, promoting the development of scientific research and clinical treatment. Currently, commonly used cell freezing solutions mostly use a single permeability protective agent such as dimethyl sulfoxide (DMSO), combined with ingredients such as fetal bovine serum (FBS). Although these ingredients can protect cells to a certain extent, high concentrations of DMSO and animal-derived serum are not allowed in the field of cell therapy. At the same time, there are problems such as low viability and poor function after cryopreservation and recovery.

[0004] Therefore, it is of great significance to develop an in vitro induced differentiation functional hepatocyte cryopreservation solution that is efficient, economical, low-toxic and meets the needs of clinical applications. Summary of the Invention

[0005] The present invention aims to overcome the problem that existing cryopreservation solutions have high DMSO content, cause significant osmotic damage and toxic damage, and do not meet clinical application requirements. The present invention provides a cryopreservation system for functional hepatocytes, comprising a balancing solution for osmotic pressure gradient balancing of cells before cryopreservation, a cryopreservation solution, and an elution solution for reducing toxic damage and osmotic damage caused by cryopreservation. The cryopreservation system does not contain animal serum.

[0006] The balanced solution includes an osmotic pressure regulator, an energy metabolism substrate, and human albumin injection;

[0007] The freezing solution includes a cryoprotectant composition, an amino acid stabilizer, and a buffer;

[0008] The elution solution includes an osmotic pressure reducer and an apoptosis inhibitor.

[0009] Furthermore, the cryoprotectant composition is a combination of at least two of dimethyl sulfoxide, propylene glycol, glycerol, polyvinyl pyrrolidone, and hydroxyethyl starch;

[0010] The amino acid stabilizer is a mixture of at least two of tryptophan, proline, and histidine;

[0011] The buffer solution is compound electrolyte injection;

[0012] The total concentration of the cryoprotectant composition is less than or equal to 10% v / v, the content of dimethyl sulfoxide is less than or equal to 5% v / v, and the total concentration of the amino acid stabilizer is 30-200 mM.

[0013] Furthermore, the electrolyte solution contains sodium ions, potassium ions and calcium ions, wherein the concentration of sodium ions is 120-150 mM, the concentration of potassium ions is 3-6 mM, and the concentration of calcium ions is 0.5-1.5 mM.

[0014] Furthermore, the osmotic pressure regulator includes mannitol and uronic acid derivatives, the concentration of mannitol is 20-50mM, and the concentration of uronic acid derivatives is 50-150mM;

[0015] Energy metabolism substrates include adenine and adenosine, with the concentration of adenine being 3-8mM and the concentration of adenosine being 2-10mM;

[0016] The human albumin is human albumin injection (20%), and the concentration of the human albumin injection (20%) is 5-40% v / v.

[0017] Furthermore, the uronic acid derivative is lactobionic acid or glucuronic acid.

[0018] Furthermore, the osmotic pressure relaxant is at least one of sucrose, trehalose and maltose, with a concentration of 150-300 mM;

[0019] Apoptosis inhibitors include mannitol, adenine, glutathione, adenosine, and lactobionic acid.

[0020] A method for cryopreservation and elution of functional hepatocytes, using a cryopreservation system for functional hepatocytes, comprises the following steps:

[0021] S1: Use the equilibrium solution to balance the cellular osmotic pressure gradient of liver parenchymal cells;

[0022] S2: Mix the equilibrated cell suspension with the freezing solution;

[0023] S3: The mixed solution is cooled to below -80°C at a rate of 0.5-2°C / min for cryopreservation;

[0024] S4: After resuscitation, the resuscitation solution is gradient eluted using the elution solution.

[0025] Furthermore, in S1, the osmotic pressure of the cells is adjusted in stages 2-3 times using the balancing solution, and the osmotic pressure change amplitude of each adjustment is not less than or equal to 100 mOsm / kg.

[0026] Furthermore, in S2, the volume mixing ratio of the cell suspension to the freezing solution is 0.5-2:1.

[0027] Furthermore, in S4, during elution, the temperature is 2-8°C and the elution time is less than 30 minutes.

[0028] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0029] 1. The present invention reduces the potential toxic damage to cells caused by a higher content of DMSO in the freezing solution and improves cell survival rate by reducing the content of DMSO or completely replacing DMSO.

[0030] 2. By optimizing the formula of the freezing solution, the present invention can better maintain the survival rate and functional status of cells during the freezing and thawing process, which is particularly important for functional cells that need to maintain high biological activity.

[0031] 3. This invention proposes a complete cryopreservation process, including three stages: pre-cryopreservation, cryopreservation, and post-cryopreservation. Each stage has specific processing steps, such as using an equilibration solution (Solution A) for cell collection and washing, using a specifically formulated cryopreservation solution (Solution B) for cryopreservation, and using an elution solution (Solution C) for post-thaw processing. This process design helps minimize cell damage and improve the quality of cells after thawing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The cell survival rate at 0 h after cryopreservation and thawing of the cryopreservation solution of Examples 1-10 of the present invention.

[0033] Figure 2 The cell survival rate 4 hours after cryopreservation and recovery in the cryopreservation solution of Examples 1-10 of the present invention.

[0034] Figure 3 This is the cell recovery rate after cryopreservation and thawing of the cryopreservation solution of Examples 1-10 of the present invention.

[0035] Figure 4 This is the characterization of the AAT secretion ability of the cryopreservation solution of Examples 1-10 of the present invention after cryopreservation and thawing.

[0036] Figure 5This is a characterization of the urea synthesis ability of the cryopreservation solutions of Examples 1-10 of the present invention after cryopreservation and thawing. DETAILED DESCRIPTION

[0037] The following will be described in more detail with reference to a schematic diagram of a cryopreservation system for functional hepatocytes and a cryopreservation elution method thereof, which shows a preferred embodiment of the present invention. It should be understood that those skilled in the art can modify the present invention described herein while still achieving the beneficial effects of the present invention. Therefore, the following description should be understood as being generally known to those skilled in the art and not as a limitation of the present invention.

[0038] The cryopreservation solution for functional hepatocytes induced to differentiate in vitro comprises a balancing solution (solution A), a cryopreservation solution (solution B), and an elution solution (solution C), and its components are as follows:

[0039] Balanced solution (Solution A): 1. Mannitol 30 mM, adenine 5 mM, glutathione 3 mM, adenosine 5 mM, lactobionic acid 100 mM, human serum albumin injection (20%) 5%, and the balance is compound electrolyte injection.

[0040] Cryopreservation solution (solution B): human albumin injection (20%) 10%-35%, dimethyl sulfoxide (DMSO) 0%-5%, propylene glycol (PG) 0%-5%, glycerol: 0%-5%, polyvinylpyrrolidone (PVP) 0%-2%, hydroxyethyl starch 3%, mannitol 30mM, adenine 5mM, glutathione 3mM, adenosine 5mM, lactobionic acid 100mM, tryptophan 50mM, proline 10mM, histidine 50mM, the balance of compound electrolyte injection

[0041] Elution solution (solution C): mannitol 30 mM, adenine 5 mM, glutathione 3 mM, adenosine 5 mM, lactobionic acid 100 mM, sucrose 200 mM, and the remainder is compound electrolyte injection.

[0042] The method of using the cryopreservation solution system for functional hepatocytes induced to differentiate in vitro is as follows: the cryopreservation process is divided into three stages: pre-cryopreservation treatment, cryopreservation and post-cryopreservation treatment. Pre-cryopreservation treatment uses a balanced solution (liquid A) to collect, wash and balance cells to help cells adapt to changes in osmotic pressure. It can reduce osmotic damage to cells during subsequent cryopreservation. During the cryopreservation process, the balanced cell suspension is mixed with the cryopreservation solution (liquid B) in a 1:1 ratio and mixed thoroughly to ensure that the cells are evenly distributed. Post-cryopreservation treatment refers to the elution treatment of the thawed cell suspension with a 4°C pre-cooled elution solution (liquid C) after recovery to reduce the toxic damage and osmotic damage of the protective agent and inhibit subsequent cell apoptosis. Specifically including the following steps:

[0043] Step 1: After cell collection, suspend the cells in culture medium to prepare a single-cell suspension and count them.

[0044] Step 2: Calculate the number of cells to be frozen based on the cell density and the capacity of the selected cryopreservation tube. The freezing density is 1×10^7 cells per ml.

[0045] Step 3: Centrifuge the cells at 500 g for 5 minutes to collect the cells.

[0046] Step 4: After collecting the cultured cell pellet, remove the supernatant from the centrifuge tube and use the balancing solution to balance the cellular osmotic pressure gradient of the hepatic parenchymal cells.

[0047] Step 5: Mix the equilibrated cell suspension with the freezing solution and gently pipette to mix to prepare a cell suspension.

[0048] Step 6: Aliquot the prepared cell suspension into labeled cryovials.

[0049] Step 7: Transfer the cryovials to a pre-cooled programmed cooling box, transfer them to a -80°C refrigerator for cooling, and transfer them to liquid nitrogen overnight for storage for one month.

[0050] Step 8: Cell Thawing: Thaw the cells quickly in a 37°C water bath. Gently shake the cryovial while thawing in the water bath. Thawing is complete when very few ice crystals remain in the tube. Remove the cryovial.

[0051] Step 9: Immediately after thawing, perform gradient elution of the resuscitated solution with elution buffer. After dilution and centrifugation, remove the supernatant and resuspend in 37°C culture medium to prepare a cell suspension. Assay cell viability and function.

[0052] The following describes the effects of freezing systems with different components on the present application through specific examples.

[0053] Example 1

[0054] An in vitro induced differentiation functional hepatocyte cryopreservation system, the cryopreservation system consisting of dimethyl sulfoxide, ethylene glycol, glycerol, human serum albumin injection, and compound electrolyte injection, wherein the component concentrations are: dimethyl sulfoxide 2% v / v, ethylene glycol 5% v / v, glycerol 3% v / v, human serum albumin injection (20%) 20% v / v, and compound electrolyte injection as the remainder.

[0055] Example 2: A freezing system for functional hepatocytes induced to differentiate in vitro, the freezing system consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, human albumin injection, polyvinyl pyrrolidone, trehalose, raffinose, hydroxyethyl starch, lactobionic acid, adenosine, glutathione, allopurinol, potassium hydroxide, potassium dihydrogen sulfate, magnesium sulfate, water for injection, etc., wherein the concentrations of the components are: dimethyl sulfoxide 2% v / v, ethylene glycol 5% v / v, glycerol 3% v / v, human albumin injection (20%) 20% v / v, polyvinyl pyrrolidone 1% w / v, trehalose 200 mM, raffinose 18 mM, hydroxyethyl starch 3%, lactobionic acid 65 mM, adenosine 3 mM, glutathione 1.8 mM, allopurinol 0.6 mM, potassium hydroxide 60 mM, potassium dihydrogen sulfate 15.5 mM, magnesium sulfate 3 mM, and the balance being water for injection.

[0056] Example 3: A freezing system for functional hepatocytes induced to differentiate in vitro, the freezing system consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, human albumin injection, polyvinyl pyrrolidone, trehalose, raffinose, hydroxyethyl starch, lactobionic acid, adenosine, glutathione, allopurinol, potassium hydroxide, potassium dihydrogen sulfate, magnesium sulfate, water for injection, etc., wherein the concentrations of the components are: dimethyl sulfoxide 4% v / v, ethylene glycol 5% v / v, glycerol 6% v / v, human albumin injection (20%) 20% v / v, polyvinyl pyrrolidone 1% w / v, trehalose 200 mM, raffinose 18 mM, hydroxyethyl starch 3%, lactobionic acid 65 mM, adenosine 3 mM, glutathione 1.8 mM, allopurinol 0.6 mM, potassium hydroxide 60 mM, potassium dihydrogen sulfate 15.5 mM, magnesium sulfate 3 mM, and the balance being water for injection.

[0057] Example 4: A cryopreservation system for functional hepatocytes induced to differentiate in vitro, the cryopreservation system comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, human serum albumin injection, polyvinyl pyrrolidone, trehalose, raffinose, hydroxyethyl starch, lactobionic acid, adenosine, glutathione, allopurinol, potassium hydroxide, potassium dihydrogen sulfate, magnesium sulfate, water for injection, etc., wherein the concentrations of the components are: dimethyl sulfoxide 3% v / v, ethylene glycol 3.33 % v / v, glycerol 6.67% v / v, human serum albumin injection (20%) 20% v / v, polyvinyl pyrrolidone 1% w / v, trehalose 200mM, raffinose 18mM, hydroxyethyl starch 3%, lactobionic acid 65mM, adenosine 3mM, glutathione 1.8mM, allopurinol 0.6mM, potassium hydroxide 60mM, potassium dihydrogen sulfate 15.5mM, magnesium sulfate 3mM, and the balance is water for injection.

[0058] Example 5: A cryopreservation system for functional hepatocytes induced to differentiate in vitro, the cryopreservation system comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, human serum albumin injection, polyvinyl pyrrolidone, trehalose, raffinose, hydroxyethyl starch, lactobionic acid, adenosine, glutathione, allopurinol, potassium hydroxide, potassium dihydrogen sulfate, magnesium sulfate, water for injection, etc., wherein the concentrations of the components are: dimethyl sulfoxide 2.5% v / v, ethylene glycol 10 % v / v, glycerol 5.67% v / v, human serum albumin injection (20%) 20% v / v, polyvinyl pyrrolidone 1% w / v, trehalose 200mM, raffinose 18mM, hydroxyethyl starch 3%, lactobionic acid 65mM, adenosine 3mM, glutathione 1.8mM, allopurinol 0.6mM, potassium hydroxide 60mM, potassium dihydrogen sulfate 15.5mM, magnesium sulfate 3mM, and the balance is water for injection.

[0059] Example 6: A freezing system for functional hepatocytes induced for differentiation in vitro, the freezing system consisting of ethylene glycol, propylene glycol, glycerol, human albumin injection, polyvinyl pyrrolidone, trehalose, raffinose, hydroxyethyl starch, lactobionic acid, adenosine, glutathione, allopurinol, potassium hydroxide, potassium dihydrogen sulfate, magnesium sulfate, water for injection, etc., wherein the concentrations of the components are: ethylene glycol 4% v / v, propylene glycol 3% v / v, glycerol 6% v / v, human albumin injection (20%) 20% v / v, polyvinyl pyrrolidone 1% w / v, trehalose 200 mM, raffinose 18 mM, hydroxyethyl starch 3%, lactobionic acid 65 mM, adenosine 3 mM, glutathione 1.8 mM, allopurinol 0.6 mM, potassium hydroxide 60 mM, potassium dihydrogen sulfate 15.5 mM, magnesium sulfate 3 mM, and the balance being water for injection.

[0060] Example 7: A freezing system for functional hepatocytes induced to differentiate in vitro, the freezing system consisting of dimethyl sulfoxide, ethylene glycol, human albumin injection, polyethylene glycol, and compound electrolyte injection, wherein the concentrations of the components are: dimethyl sulfoxide 3% v / v, ethylene glycol 10% v / v, human albumin injection (20%) 25% v / v, polyethylene glycol 2.5%, and the balance compound electrolyte injection.

[0061] Example 8: A freezing system for functional hepatocytes induced to differentiate in vitro, the freezing system consisting of dimethyl sulfoxide, ethylene glycol, human albumin injection, polyethylene glycol, and compound electrolyte injection, wherein the concentrations of the components are: dimethyl sulfoxide 4.8% v / v, ethylene glycol 2.7% v / v, human albumin injection (20%) 25% v / v, polyethylene glycol 3%, and the balance compound electrolyte injection.

[0062] Example 9: A freezing system for functional hepatocytes induced to differentiate in vitro, the freezing system consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, human albumin injection, dextran 40, proline, and compound electrolyte injection, wherein the concentrations of the components are: dimethyl sulfoxide 4.8% v / v, ethylene glycol 6.7% v / v, propylene glycol 7.2% v / v, glycerol 5% v / v, human albumin injection (20%) 25% v / v, dextran 40 5% v / v, and compound electrolyte injection as the remainder.

[0063] Example 10: A freezing system for functional hepatocytes induced for differentiation in vitro, the freezing system consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, human serum albumin injection, proline, and compound electrolyte injection, wherein the concentrations of the components are: dimethyl sulfoxide 4% v / v, ethylene glycol 3.5% v / v, propylene glycol 6% v / v, glycerol 4% v / v, human serum albumin injection (20%) 25% v / v, proline 40 mM, glycine 80 mM, and the remainder of compound electrolyte injection.

[0064] like Figure 1-5 As shown, through in-depth analysis of Examples 1-10, we found that the cryopreservation solutions of Examples 2 and 3 performed particularly well. When performing cryopreservation and thawing experiments on functional hepatocytes induced and differentiated in vitro, these two groups of cryopreservation solutions were able to maintain a relatively high cell viability and recovery rate. More importantly, the cells after cryopreservation and thawing were still able to retain the ability to secrete AAT and synthesize urea. This fully demonstrates that the cryopreservation solution of the present invention can not only maintain the viability of functional hepatocytes induced and differentiated in vitro after cryopreservation, but also effectively maintain the function of the cells, greatly reducing the damage to the cells caused by the cryopreservation process, and achieving high-quality preservation of functional hepatocytes induced and differentiated in vitro, providing strong support for research and application in related fields.

[0065] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A freezing system for functional hepatocytes, characterized in that: The invention comprises a balancing solution for balancing the osmotic pressure gradient of cells before freezing, a freezing solution, and an elution solution for reducing toxic damage and osmotic damage caused by freezing, wherein the freezing system does not contain animal serum; The balanced solution includes an osmotic pressure regulator, an energy metabolism substrate and human serum albumin injection; The freezing solution includes a cryoprotectant composition, an amino acid stabilizer and a buffer; The elution solution includes an osmotic pressure relaxant and a cell apoptosis inhibitor.

2. The cryopreservation system for functional hepatocytes according to claim 1, characterized in that: The cryoprotectant composition is at least two of dimethyl sulfoxide, propylene glycol, glycerol, polyvinyl pyrrolidone, and hydroxyethyl starch; The amino acid stabilizer is a mixture of at least two of tryptophan, proline, and histidine; The buffer solution is a compound electrolyte injection; The total concentration of the cryoprotectant composition is less than or equal to 10% v / v, the content of the dimethyl sulfoxide is less than or equal to 5% v / v, and the total concentration of the amino acid stabilizer is 30-200 mM.

3. The freezing system for functional hepatocytes according to claim 1, characterized in that: The electrolyte solution contains sodium ions, potassium ions and calcium ions, wherein the concentration of sodium ions is 120-150 mM, the concentration of potassium ions is 3-6 mM, and the concentration of calcium ions is 0.5-1.5 mM.

4. The freezing system for functional hepatocytes according to claim 1, characterized in that: The osmotic pressure regulator includes mannitol and uronic acid derivatives, the concentration of the mannitol is 20-50mM, and the concentration of the uronic acid derivative is 50-150mM; The energy metabolism substrates include adenine and adenosine, the concentration of adenine is 3-8 mM, and the concentration of adenosine is 2-10 mM; The human albumin is human albumin injection (20%), and the concentration of the human albumin injection (20%) is 5-40% v / v.

5. The freezing system for functional hepatocytes according to claim 4, characterized in that: The uronic acid derivative is lactobionic acid or glucuronic acid.

6. The freezing system for functional hepatocytes according to claim 1, characterized in that: The osmotic pressure moderating agent is at least one of sucrose, trehalose and maltose, with a concentration of 150-300 mM; The cell apoptosis inhibitors include mannitol, adenine, glutathione, adenosine, and lactobionic acid.

7. A method for cryopreservation and elution of functional hepatocytes, comprising: using the cryopreservation system for functional hepatocytes according to any one of claims 1 to 6, wherein: The following steps are involved: S1: Use the equilibrium solution to balance the cellular osmotic pressure gradient of liver parenchymal cells; S2: Mix the equilibrated cell suspension with the freezing solution; S3: The mixed solution is cooled to below -80°C at a rate of 0.5-2°C / min for cryopreservation; S4: After resuscitation, the resuscitation solution is gradient eluted using the elution solution.

8. The method for cryopreservation and elution of functional hepatocytes according to claim 7, characterized in that: In S1, the osmotic pressure of the cells is adjusted in stages 2-3 times using the balancing solution, and the osmotic pressure change amplitude of each adjustment is not less than or equal to 100 mOsm / kg.

9. The method for cryopreservation and elution of functional hepatocytes according to claim 7, characterized in that: In the S2, the volume mixing ratio of the cell suspension to the freezing solution is 0.5-2:

1.

10. The method for cryopreservation and elution of functional hepatocytes according to claim 7, characterized in that: In said S4, during elution, the temperature is 2-8°C and the elution time is less than 30 minutes.

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