3D preparation method for hepatocytes

Abstract

Disclosed in the present invention are a 3D preparation method for and a use of hepatocytes. In the present invention, hepatic precursor cells are differentiated into functional hepatocytes via low-oxygen conditions and a specific culture medium, and after sampling and counting, hepatic spheroids are subjected to liquid nitrogen cryopreservation. The 3D preparation of hepatocytes does not require a microcarrier, and digestive enzyme digestion is not needed.

Description

A 3D preparation method for hepatocytes Technical Field

[0001] The present application relates to the field of cell engineering technology, and in particular to a 3D preparation method for liver cells. Background Art

[0002] China is the world's leading country for liver disease. Over one million new cases of liver failure occur in my country each year. Liver transplantation is the only curative option for patients with liver failure. However, the extreme shortage of liver sources significantly limits the application of this treatment option, and the majority of patients with liver failure die from liver failure while waiting for a liver donor. Bioartificial livers offer a potential treatment option for liver failure. However, functional mature hepatocytes rarely proliferate, significantly limiting their clinical application. Therefore, the current bottleneck in the clinical application of bioartificial liver technology is obtaining hepatocytes with improved functionality that can be produced in large quantities. Mature hepatocytes secrete albumin, adsorb and detoxify bilirubin and blood ammonia, and promote hepatocyte self-renewal, thereby reshaping the liver's regenerative microenvironment and promoting liver regeneration. Current methods for producing hepatocytes primarily rely on adherent 2D culture under normoxic conditions, which exhibits lower cell viability and biological function than 3D cultured cells. Conventional 3D cultured cells require the use of microcarriers for expansion, which is costly, complex, and prone to exogenous viral contamination. Technical issues

[0003] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide a 3D preparation method and application of hepatocytes. Technical Solutions

[0004] To achieve the above objectives, the technical solutions adopted in this application are:

[0005] In a first aspect, the present application provides a 3D preparation method for hepatocytes, comprising the following steps:

[0006] S1. Inoculate hepatic progenitor cells into 3D culture;

[0007] S2. A mixed gas containing 1% to 5% O2 was introduced into the 3D suspension device, and the hepatic progenitor cells were cultured in a serum-free culture medium for 96 hours to obtain cell spheres, which were then centrifuged and the supernatant removed.

[0008] S3. Add hepatocyte culture medium to the 3D suspension device and maintain hypoxic conditions with a mixed gas of 1% to 5% O2. Continue suspension culture for 6 days, changing the medium every other day. After 6 days, centrifuge to remove the supernatant and freeze.

[0009] Furthermore, the hepatic progenitor cells include at least one of pig hepatic progenitor cells, adult hepatic progenitor cells, and hepatic progenitor cells differentiated from embryonic stem cells.

[0010] Preferably, the hepatic progenitor cells are hepatic progenitor cells differentiated from embryonic stem cells. Furthermore, the passage density of the hepatic progenitor cells is 5×10 4 cells to 2×10 5 cells.

[0011] Furthermore, in the culture system, the dosage of the hepatic progenitor cells is: 5×10 4 cells / mL to 2×10 5 cells / mL.

[0012] Furthermore, the mixed gas is a mixed gas of 94% by volume N2, 5% by volume CO2 and 1% by volume O2.

[0013] Furthermore, in step S2, the serum-free culture medium for hepatic progenitor cells includes the following components at the following concentrations: DMEM basal culture medium, B27 with a final concentration of 1wt%~4wt%, KSR with a final concentration of 1wt%~5wt%, CHIR with a final concentration of 1 μM~3μM, EGF with a final concentration of 10 ng / mL~30 ng / mL, L-glutamine with a final concentration of 1.5mmol / ml~2.5mmol / ml, vitamin C with a final concentration of 45μg / ml~55μg / ml, and Heparin with a final concentration of 50 μg / mL~150 μg / mL.

[0014] Furthermore, in step S3, the hepatocyte serum-free culture medium includes the following components at the following concentrations: William E basal medium, hepatocyte growth supplement (HGS, Cat.No.5252) with a final concentration of 5wt%-10wt%, ITS with a final concentration of 1wt%-2wt%, GlutaMAX with a final concentration of 1wt%-2wt%, Heparin with a final concentration of 50 μg / mL~150 μg / mL, HEPES with a final concentration of 5wt%-10wt%, dexamethasone with a final concentration of 0.1wt%-1wt%, and BSA with a final concentration of 1wt%-5wt%.

[0015] In a second aspect, the present application provides hepatocytes prepared by the above method. Beneficial effects

[0016] Compared with existing technologies, this application has the following advantages: It provides a 3D preparation method and application of hepatocytes. This application seeded hepatic progenitor cells, differentiated them into functional hepatocytes under hypoxic conditions and a specific culture medium, then sampled and counted them, and the hepatocyte spheres were cryopreserved in liquid nitrogen. The 3D preparation of hepatocytes in this application does not require microcarriers or digestion enzymes, is simple to operate, and is more functional than hepatocytes prepared under 2D conditions, making it more beneficial for clinical treatment. Best Mode for Carrying Out the Invention

[0017] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0019] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0020] Example 1

[0021] A 3D preparation method for hepatocytes, comprising the following steps:

[0022] (1) Single hepatic progenitor cells, whose cell phenotypes were identified by flow cytometry, were inoculated into a 125 mL 3D suspension culture spinner flask at a number of 2.5×106 viable cells. 50 mL of serum-free medium for hepatic progenitor cells was added to the spinner flask. The rotation speed was set to 35 rpm. Under the mixed gas conditions of 94% N2, 5% CO2, and 1% O2 (hypoxic conditions), 3D culture was continued for 48 h at 37 °C. Then, 25 mL of serum-free medium for hepatic progenitor cells was added to the spinner flask, the rotation speed was adjusted to 45 rpm, and hypoxic culture was continued for 48 h to obtain hepatic progenitor cell spheres.

[0023] (2) Centrifuge the hepatic precursor cell sphere suspension in the spinner flask at 300g for 5 minutes, remove the supernatant, and then add 75mL of hepatocyte serum-free medium, resuspend and transfer to the spinner flask. Continue to culture in a mixed gas of 94% N2, 5% CO2 and 1% O2 (hypoxic conditions) at a speed of 45 rpm for 48 hours. The culture temperature is 37℃. Then continue to collect the cell sphere suspension, centrifuge to remove the supernatant, add fluid, and continue to culture in the hypoxic spinner flask for 48 hours. Repeat the above steps to continue to collect the cell sphere suspension, centrifuge to remove the supernatant, add fluid, and continue to culture in the hypoxic spinner flask for 48 hours.

[0024] (3) Transfer the hepatocyte suspension after preparation to a 250 mL centrifuge bottle, take 1 mL of the suspension, centrifuge at 300 g for 5 minutes, collect the supernatant for subsequent albumin secretion Elisa detection, then add 1 mL of Tryple lysis buffer to the precipitate, blow it evenly, and place it in a 37-degree water bath for digestion for 15 minutes to 20 minutes. During this period, use a pipette to blow 5-10 times every 10 minutes to accelerate lysis. After the hepatocyte spheres are completely lysed, use an NC-200 counter to calculate the cell viability and cell number in the suspension, and use this to calculate the cell number and viability obtained in the preparation. The flow cytometric identification of cell phenotypes of hepatocytes is shown in Figure 2.

[0025] (4) Centrifuge the 250 mL centrifuge bottle at 300 g for 5 min, remove the supernatant, and add the calculated volume of CS10 freezing solution to the precipitate at a ratio of 2 × 107 viable cells / mL freezing solution according to the above counting results. Blow it evenly with a pipette and add it to the freezing straw or freezing bag. Then place it in a programmed cooling device for programmed cooling. After cooling, transfer it to a liquid nitrogen tank for long-term freezing.

[0026] (5) The serum-free culture medium for hepatic progenitor cells includes the following components at the following concentrations: DMEM basal culture medium, B27 with a final concentration of 1wt%~4wt%, KSR with a final concentration of 1wt%~5wt%, CHIR with a final concentration of 1 μM~3μM, EGF with a final concentration of 10 ng / mL~30 ng / mL, L-glutamine with a final concentration of 1.5mmol / ml~2.5mmol / ml, vitamin C with a final concentration of 45μg / ml~55μg / ml, and Heparin with a final concentration of 50 μg / mL~150 μg / mL.

[0027] (6) The serum-free culture medium for hepatocytes comprises the following components at the following concentrations: William E basal medium, hepatocyte growth supplement (HGS, Cat. No. 5252) with a final concentration of 5 wt%-10 wt%, ITS with a final concentration of 1 wt%-2 wt%, GlutaMAX with a final concentration of 1 wt%-2 wt%, Heparin with a final concentration of 50 μg / mL~150 μg / mL, HEPES with a final concentration of 5 wt%-10 wt%, dexamethasone with a final concentration of 0.1 wt%-1 wt%, and BSA with a final concentration of 1 wt%-5 wt%.

[0028] Test results showed that hepatic progenitor cells highly expressed cell markers such as CK19 and AFP, with a positive rate exceeding 95%, meeting industry requirements for the identification of hepatic progenitor cells. Hepatocytes highly expressed cell markers such as ALB, with a positive rate exceeding 80%, meeting industry requirements for the identification of hepatocytes. Hepatocytes prepared under 3D hypoxia secreted higher amounts of albumin than hepatocytes prepared under other conditions.

[0029] Comparative Example 1: Compared to Example 1, 3D normoxic culture conditions were employed: the incubator environment was a mixed gas atmosphere of 95% air and 5% CO₂ (normoxia). The remaining steps, parameters, and reagents were the same as in Example 1 to obtain hepatocytes.

[0030] Comparative Example 2: Compared to Example 1, 2D hypoxic culture conditions were employed: 2.5×10⁶ single hepatocyte progenitor cells, whose cell phenotypes had been verified by flow cytometry, were seeded at a density of 3.3×10⁴ cells / cm⁻ in a T75 culture flask for expansion. 75 mL of serum-free hepatocyte progenitor cell culture medium was added to the flask. 2D culture was continued for 96 hours at 37°C under a mixed gas atmosphere of 94% N₂, 5% CO₂, and 1% O₂ (hypoxic conditions). The supernatant was then removed, and the flask was supplemented with 75 mL of serum-free hepatocyte culture medium. Hypoxic culture was continued for 48 hours, followed by a 75 mL change of the medium. Hypoxic culture was continued for another 48 hours, followed by another 75 mL change of the medium. One mL of supernatant was collected for subsequent albumin secretion ELISA testing. The supernatant was removed, and the cells were digested using Tryple digestion solution, a pancreatic enzyme substitute. After centrifugation and supernatant removal, the cells were washed twice with DPBS solution to obtain 2D cultured hepatocytes. The components of the serum-free medium for hepatic progenitor cells and the serum-free medium for hepatocytes were the same as those in Example 1.

[0031] Comparative Example 3: Compared to Example 1, 2D normoxic culture conditions were employed: 2.5×10⁶ single hepatocyte progenitor cells, whose cell phenotypes had been verified by flow cytometry, were seeded at a density of 3.3×10⁴ cells / cm⁻ in a T75 culture flask for expansion. 75 mL of serum-free hepatocyte progenitor cell culture medium was added to the flask. 2D culture was continued for 96 hours at 37°C in a mixture of 95% air and 5% CO₂ (normoxia). The supernatant was then removed, and the flask was supplemented with 75 mL of serum-free hepatocyte culture medium. Normoxia continued for 48 hours, followed by a 75 mL change of the medium. Normoxia continued for another 48 hours, followed by another 75 mL change of the medium. One mL of supernatant was collected for subsequent albumin secretion ELISA testing. The supernatant was removed, and the cells were digested using Tryple digestion solution, a pancreatic enzyme substitute. After centrifugation and supernatant removal, the cells were washed twice with DPBS solution to obtain 2D cultured hepatocytes. The components of the serum-free medium for hepatic progenitor cells and the serum-free medium for hepatocytes were the same as those in Example 1.

[0032] Example 2: Application of hepatocytes in artificial liver support system for the treatment of acute liver failure. The total number of cells was about 1×10 10 Remove the cryovial or bag of hepatocytes from the liquid nitrogen tank and thaw in a 37°C water bath for approximately 3 minutes. Transfer the entire hepatocyte sphere suspension (prepared in Example 1) in the cryovial or bag to a 500 mL centrifuge bottle and mix thoroughly by inversion. Then, remove a 1 mL sample and centrifuge at 300 g for 5 minutes. Remove the supernatant and add 1 mL of Tryple lysis buffer to the pellet. After vortexing, place the pellet in a 37°C water bath for 15-20 minutes. Pipette 5-10 times every 10 minutes to accelerate lysis. After complete lysis of the hepatocyte spheres, count the viability and number of cells in the suspension using an NC-200 counter. This will be used to calculate the number and viability of recovered cells.

[0033] A 500mL centrifuge bottle containing the suspension of hepatocyte spheres was centrifuged at 180g for 5 minutes, the supernatant was removed, and the cells were washed twice with normal saline. Then, 1L of normal saline + 5% human albumin was added to prepare the final preparation. The preparation was then poured into a sterile bioreactor and placed in a bioartificial liver support system. Bioartificial liver treatment was performed on patients with liver failure in vitro for 6 hours. Hepatocytes in the artificial liver support system can effectively play a role in scavenging toxins, thereby promoting the self-repair of liver function and thus playing a therapeutic role in liver failure.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application. Industrial Applicability

[0035] The 3D preparation method of hepatocytes described in the present invention does not require microcarriers or digestion with digestive enzymes, is simple to operate, has stronger functions than hepatocytes prepared under 2D conditions, and is more conducive to clinical treatment.

Claims

1. A 3D preparation method of hepatocytes, characterized in that, it includes the following steps: S1. Inoculate and perform 3D culture on hepatic progenitor cells; S2. Introduce a mixed gas containing O with a concentration of 1% - 5% into the 3D suspension device, continuously culture the hepatic progenitor cells in a serum-free medium for hepatic progenitor cells for 96 h to obtain cell spheres, and centrifuge to remove the supernatant; 2 ​ S3. Add hepatocyte culture medium to the 3D suspension device, maintain a hypoxic condition with a mixed gas of 1% - 5% O 2 , and continue suspension culture for 6 days. Replace the culture medium every other day. After 6 days, centrifuge to remove the supernatant and freeze it.

2. The 3D preparation method of hepatocytes according to claim 1, characterized in that, the hepatic progenitor cells include at least one of porcine hepatic progenitor cells, adult hepatic progenitor cells, and hepatic progenitor cells differentiated from embryonic stem cells.

3. The 3D preparation method of hepatocytes according to claim 2, characterized in that, the hepatic progenitor cells are preferably hepatic progenitor cells differentiated from embryonic stem cells.

4. The 3D preparation method of hepatocytes according to claim 1, characterized in that, The passage density of the hepatic progenitor cells is 5×10 4 cells ~ 2×10 5 cells.

5. The 3D preparation method of hepatocytes according to claim 1, characterized in that, In the culture system, the dosage of the hepatic progenitor cells is: 5×10 4 cells / mL to 2×10 5 cells / mL.

6. The 3D preparation method of hepatocytes according to claim 1, characterized in that, The mixed gas is N with a volume concentration of 94% 2 , CO with a volume concentration of 5% 2 and O with a volume concentration of 1% 2 mixed gas.

7. The 3D preparation method of hepatocytes according to claim 1, characterized in that, in the step S2, the serum-free medium for hepatic progenitor cells includes components with the following concentrations: DMEM basal medium, B27 with a final concentration of 1 wt% - 4 wt%, KSR with a final concentration of 1 wt% - 5 wt%, CHIR with a final concentration of 1 μM - 3 μM, EGF with a final concentration of 10 ng / mL - 30 ng / mL, L-glutamine with a final concentration of 1.5 mmol / ml - 2.5 mmol / ml, vitamin C with a final concentration of 45 μg / ml - 55 μg / ml, and Heparin with a final concentration of 50 μg / mL - 150 μg / mL.

8. The 3D preparation method of hepatocytes according to claim 1, characterized in that, in the step S3, the serum-free medium for hepatocytes includes components with the following concentrations: William E basal medium, hepatocyte growth supplement (HGS, Cat.No.5252) with a final concentration of 5 wt% - 10 wt%, ITS with a final concentration of 1 wt% - 2 wt%, GlutaMAX with a final concentration of 1 wt% - 2 wt%, Heparin with a final concentration of 50 μg / mL - 150 μg / mL, HEPES with a final concentration of 5 wt% - 10 wt%, dexamethasone with a final concentration of 0.1 wt% - 1 wt%, and BSA with a final concentration of 1 wt% - 5 wt%.

9. Hepatocytes obtained by the preparation method according to any one of claims 1 - 6.