Culture system for in-vitro amplification of mouse bone marrow hematopoietic stem cells and preparation method

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a culture system for in-vitro amplification of mouse bone marrow hematopoietic stem cells and a preparation method. In order to solve the restrictive problem of HSCT, the culture system takes a DMEM / F12 culture medium as a basic culture medium, ITS-X, P / S / G, a TPO trehalose solution, an SCF trehalose solution and a tremella polysaccharide aqueous solution are added, the volume fractions of ITS-X and P / S / G in the whole culture system are 1%, the concentration of TPO trehalose is 100 ng / mL, the concentration of SCF trehalose is 10 ng / mL, and the mass percent concentration of tremella polysaccharide is 0.001%.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a culture system and a preparation method for in vitro expansion of mouse bone marrow hematopoietic stem cells. Background Art

[0002] Hematopoietic stem cells (HSCs) are generally considered to be the basis of the adult hematopoietic system. They have the important characteristics of long-term self-renewal and the production of all mature blood cell lineages during the life cycle of an organism. They can establish the body's hematopoietic system and immune system, and have broad application prospects in the treatment of various blood diseases and immunodeficiency diseases. HSCs can not only maintain hematopoietic function by self-renewal to produce new HSCs, but also multidirectionally differentiate into all types of functional blood cells to maintain hematopoietic function. In 2000, Irving Weissman et al. of Stanford University first proposed the hematopoietic hierarchy "pyramid" model of HSCs from multipotential to unipotential and finally mature. Long-term self-renewing hematopoietic stem cells (long-term HSCs, LT-HSCs) with self-renewal and long-term hematopoietic reconstruction capabilities are located at the top of the model. They have uninterrupted self-renewal capabilities, regulate the body to maintain the required homeostasis and continuous blood cell circulation throughout its life, and produce short-term self-renewing hematopoietic stem cells (short-term HSCs, ST-HSCs) with short-term multilineage reconstruction capabilities. ST-HSC has short-term transplantation ability and can quickly restore the recipient's hematopoietic system 8-12 weeks after transplantation. ST-HSC also has high proliferation characteristics and can differentiate into multipotent progenitors (MPP). MPP has stronger proliferation characteristics, but it does not have the ability to self-renew. In addition, ST-HSC and MPP can eventually differentiate into different types of blood cells, such as T lymphocytes, B lymphocytes, natural killer cells (NK cells), platelets, red blood cells, monocytes, dendritic cells (DC cells) and macrophages.

[0003] Although HSCs are responsible for the production of billions of hematopoietic cells every day, genetic mutations in this process can lead to leukemia, myelodysplasia, and bone marrow failure. Although hematopoietic diseases were once almost fatal, hematopoietic stem cell transplantation (HSCT) now offers hope for hematopoietic diseases. HSCT has important curative potential in the treatment of hematological malignancies, autoimmune diseases, genetic diseases, and correction of severe defects in the hematopoietic system. It is also the final treatment option for patients with various cancers. In the current allogeneic hematopoietic stem cell transplantation (allo-HSCT) model, the transplantation success rate for malignant and non-malignant diseases is close to 100%, but graft-versus-host disease, severe infection caused by poor immune system reconstitution, and disease relapse are still the main causes of transplant failure; and in autologous hematopoietic stem cell transplantation (auto-HSCT), relapse is still the most common cause of death. Although the main sources of cells for HSCT in clinical practice today are bone marrow, mobilized peripheral blood, and neonatal umbilical cord blood, there are still some clinical problems that need to be solved.

[0004] The main problem hindering the development of HSC therapy is that the number of HSC extracted from the donor is far from meeting the requirements of clinical application. The number of available donor hematopoietic stem cells is often insufficient for bone marrow reconstruction, so people are gradually turning their attention to the large-scale expansion of HSC in vitro. The key to the success of HSCT depends on the number of donor HSC and the efficiency of transplantation. Therefore, the effective expansion of HSC in vitro has become an important means to solve the problem and is also one of the current research hotspots in the HSC field.

[0005] The in vitro expansion method of HSC is mainly achieved by simulating the bone marrow microenvironment of HSC. Stem cells need to reside in the bone marrow to maintain their stemness. Based on the simulation of the bone marrow microenvironment, the in vitro expansion method of HSC mainly includes two categories: stromal cell co-culture method and free cytokine addition method. Among them, the stromal cell co-culture method utilizes the important regulatory role of bone marrow stromal cells in the microenvironment on HSC self-renewal, differentiation and mobilization, and co-cultures stromal cells with HSC as supporting cells during the in vitro expansion of HSC. These stromal cells can regulate the fate and function of HSC by directly contacting HSC and secreting cytokines and extracellular matrix. The free cytokine addition method directly adds cytokines for HSC culture by utilizing the support and promotion of cytokines on HSC growth. Compared with the stromal cell co-culture method, the free cytokine addition method can reduce the introduction of exogenous cells and the culture process is simple, so it has become the most commonly used in vitro expansion method of HSC.

[0006] Traditional cell culture media are mostly a combination of basal culture media and fetal bovine serum. Although fetal bovine serum is rich in growth factors required for in vitro cell culture and is suitable for various types of cell culture, it has many disadvantages for in vitro HSC expansion and clinical application, such as the uncertain composition of fetal bovine serum, which is not suitable for repeated experiments; the threat of immune response and blood-borne diseases caused by heterologous serum; microbial contamination (viruses, bacteria, endotoxins, etc.). Therefore, serum-free culture media are often used for in vitro expansion of HSC, or serum analogs are added.

[0007] Therefore, there is an urgent need to establish an efficient and high-quality HSC in vitro expansion system in this field to effectively solve the limiting problems of HSCT, benefit patients from the disease itself, economy, psychology and other aspects, and achieve good social benefits. It is mainly reflected in the following points: ① It reduces the need for human leukocyte antigen (HLA) matching, a product of the human major histocompatibility complex, thereby extending the effectiveness of transplantation to almost all patients, so that 30%-40% of patients who currently lack HLA-matched donors can obtain transplantation opportunities, which is conducive to alleviating the serious donor shortage problem currently faced by patients waiting for transplantation in clinical practice; ② It reduces the recurrence rate of malignant primary diseases after transplantation and the risk of chronic graft-versus-host disease, and also reduces the high medical expenses generated thereby, improves the long-term quality of life of transplant patients, and reduces the social medical burden; ③ While expanding progenitor cells, maintaining and expanding LT-HSC not only promotes neutrophil engraftment and early hematopoietic reconstruction, but also reduces the risk of infection after transplantation and improves the success rate of transplantation. Summary of the invention

[0008] In view of the above problems, the present invention provides a culture system and a preparation method for in vitro expansion of mouse bone marrow hematopoietic stem cells.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The invention provides a culture system for in vitro expansion of mouse bone marrow hematopoietic stem cells. The culture system uses a DMEM / F12 culture medium as a basic culture medium, and is added with ITS-X, P / S / G, a TPO trehalose solution, an SCF trehalose solution and a tremella polysaccharide aqueous solution. The volume fractions of ITS-X and P / S / G in the entire culture system are both 1%, the concentration of TPO trehalose is 100 ng / mL, the concentration of SCF trehalose is 10 ng / mL, and the mass percentage concentration of tremella polysaccharide is 0.001%.

[0011] Furthermore, the concentration of the TPO trehalose solution is 10 μg / mL, the concentration of the SCF trehalose solution is 1 μg / mL, and the concentration of the Tremella polysaccharide aqueous solution is 20 mg / mL.

[0012] The mass percentage concentration of 0.001% means: if the total volume of the culture medium is 50 mL, then 0.0005 g, i.e. 0.5 mg, is added, and the concentration is 20 mg / mL, then the added volume is 0.025 mL, i.e. 25 μL.

[0013] The present invention also provides a method for preparing a culture system for in vitro expansion of mouse bone marrow hematopoietic stem cells, comprising the following steps:

[0014] Step 1, dissolve 5 g Tremella polysaccharide powder in 250 mL sterile water, shake vigorously, and configure to 20 mg / mL for standby use;

[0015] Step 2, first dissolve 10 μg SCF powder in 10 μL sterile water, mix well, and prepare to 1 mg / mL; then add 10 μL of the 1 mg / mL mixture to 90 μL of a 5% trehalose solution to prepare to 0.1 mg / mL; then add 100 μL of the 0.1 mg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 10 μg / mL; finally, add 100 μL of the 10 μg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 1 μg / mL; package in 500 μL tubes, store at -80°C for future use;

[0016] Step 3, first dissolve 100 μg TPO powder in 200 μL sterile water, mix well, and prepare to 0.5 mg / mL; then add 200 μL of 0.5 mg / mL mixed solution to 800 μL of 5% trehalose solution by mass fraction to prepare to 0.1 mg / mL; finally, add 100 μL of 0.1 mg / mL mixed solution to 900 μL of 5% trehalose solution by mass fraction to prepare to 10 μg / mL; package in 500 μL tubes, store at -80°C for future use;

[0017] Step 4, directly package P / S / G and ITS-X into 500 μL tubes and store at -80°C for future use;

[0018] Step 5, add 500 μL P / S / G, 500 μL ITSX, 500 μL 1 μg / mL SCF in step 2, 500 μL 10 μg / mL TPO in step 3, and 25 μL 20 mg / mL Tremella polysaccharide in step 1 to 47.975 mL DMEM / F12 medium to prepare a serum-free Tremella polysaccharide medium with a mass percentage concentration of 0.001%.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. No fetal bovine serum or protein analogs are added to this culture system, which avoids the uncertainty of the composition of fetal bovine serum and is not suitable for repeated experiments; there are threats of immune response and blood-borne diseases caused by heterologous serum; microbial contamination (viruses, bacteria, endotoxins, etc.), etc.

[0021] 2. Tremella fuciformis polysaccharide (TFP) is not only economical, but also can provide a three-dimensional spatial structure for the growth of HSC, meeting the conditions of a carrier molecule.

[0022] 3. HSC is very sensitive to redox status. Reactive oxygen can cause HSC depletion, while TFP has antioxidant activity and can maintain the growth of HSC.

[0023] 4. TFP promoted the increase in the number of cultured cells and the proportion of HSC and LT-HSC in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Mouse bone marrow CD117 + Cell morphology of each group under microscope after 0 and 9 days of cell expansion in vitro (400×).

[0025] Figure 2 Mouse bone marrow CD117 +The cell expansion multiples of each group after 0, 5, 7, and 9 days of in vitro expansion. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0026] Figure 3 Mouse bone marrow CD117 + The proportion of each group of cells (HSC, LT-HSC, ST-HSC, MPP) after 0 and 9 days of cell expansion in vitro, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001. DETAILED DESCRIPTION

[0027] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0028] Example 1

[0029] The culture system for in vitro expansion of mouse bone marrow hematopoietic stem cells of the present embodiment uses DMEM / F12 culture medium as the basal culture medium, and adds ITS-X, P / S / G, TPO trehalose solution, SCF trehalose solution and Tremella polysaccharide aqueous solution. The volume fractions of ITS-X and P / S / G in the entire culture system are both 1%, the concentration of TPO trehalose is 100 ng / mL, the concentration of SCF trehalose is 10 ng / mL, and the mass percentage concentration of Tremella polysaccharide is 0.001%; ​​the concentration of the TPO trehalose solution is 10 μg / mL, the concentration of the SCF trehalose solution is 1 μg / mL, and the concentration of the Tremella polysaccharide aqueous solution is 20 mg / mL.

[0030] The preparation method of the culture system comprises the following steps:

[0031] Step 1, dissolve 5 g Tremella polysaccharide powder in 250 mL sterile water, shake vigorously, and configure to 20 mg / mL for standby use;

[0032] Step 2, first dissolve 10 μg SCF powder in 10 μL sterile water, mix well, and prepare to 1 mg / mL; then add 10 μL of the 1 mg / mL mixture to 90 μL of a 5% trehalose solution to prepare to 0.1 mg / mL; then add 100 μL of the 0.1 mg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 10 μg / mL; finally, add 100 μL of the 10 μg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 1 μg / mL; package in 500 μL tubes, store at -80°C for future use;

[0033] Step 3, first dissolve 100 μg TPO powder in 200 μL sterile water, mix well, and prepare to 0.5 mg / mL; then add 200 μL of 0.5 mg / mL mixed solution to 800 μL of 5% trehalose solution by mass fraction to prepare to 0.1 mg / mL; finally, add 100 μL of 0.1 mg / mL mixed solution to 900 μL of 5% trehalose solution by mass fraction to prepare to 10 μg / mL; package in 500 μL tubes, store at -80°C for future use;

[0034] Step 4, directly package P / S / G and ITS-X into 500 μL tubes and store at -80°C for future use;

[0035] Step 5, add 500 μL P / S / G, 500 μL ITSX, 500 μL 1 μg / mL SCF in step 2, 500 μL 10 μg / mL TPO in step 3, and 25 μL 20 mg / mL Tremella polysaccharide in step 1 to 47.975 mL DMEM / F12 medium to prepare a serum-free Tremella polysaccharide medium with a mass percentage concentration of 0.001%.

[0036] Example 2

[0037] 1. Isolation and extraction of mouse bone marrow cells

[0038] (1) Prepare sterile scissors, tweezers, culture dishes, PBS, gauze, 1 mL syringe, 5 mL syringe, 200 mesh cell filter, etc. in advance.

[0039] (2) Mice were killed by myelotomy and soaked in 75% alcohol for later use.

[0040] (3) Fix the mouse on the operating table, cut the skin from the abdomen to the limbs, peel off the skin to expose the limbs, cut as much muscle as possible, clamp the metatarsal bones, and pull out the ilium-femur-tibia with a little force upward (be careful to maintain integrity), place it in a sterilized culture dish filled with sterile PBS, and keep the dish on ice until use.

[0041] (4) According to step (3), the limb bones of the mouse are peeled off and placed in a dish, and placed on ice for later use.

[0042] (5) Wrap the bone with a double layer of sterile gauze and carefully remove the muscle tissue.

[0043] (6) Soak the separated bones in another sterilized culture dish containing sterile PBS and move it into the clean bench for subsequent steps.

[0044] (7) Use a 5 mL sterile syringe to absorb sterile PBS, install a 1 mL syringe needle, use scissors to cut off the epiphyseal ends on both sides of the bone, find the bone hole and gently insert it into the bone marrow cavity, and use PBS to flush out the bone marrow cells. Generally, flush the bone marrow cavity three times (once in the forward direction, once in the reverse direction, and once in the forward direction). After flushing, the bone marrow cavity can be seen to be white.

[0045] (8) Resuspend the collected bone marrow cells by repeated pipetting, filter through a 200-mesh cell strainer, and centrifuge at 450 g, 4°C, for 10 min.

[0046] (9) Discard the supernatant, add an appropriate amount of red blood cell lysis buffer, shake to mix, and incubate on ice for 15 min.

[0047] (10) Centrifuge at 450 g, 4°C, for 10 min, discard the supernatant, resuspend the cells in an appropriate amount of PBS, count the live cells, and place on ice until use.

[0048] 2. Mouse bone marrow CD117 + Enrichment of cells

[0049] (1) According to the number of cells counted in the above steps, the cell concentration was adjusted to 10 7 cells / 80μL PBS, at a rate of 10 7 Add 20 μL CD117 MicroBeads to the cells, mix thoroughly by pipetting, and then incubate the cell suspension at 4°C in the dark for 15 min.

[0050] (2) After the incubation, the cell suspension was incubated at 10 7 Add 1 mL of PBS to the cells, mix the cells, and centrifuge at 450 g, 4°C, for 10 min.

[0051] (3) Discard the supernatant and separate the cell pellets according to the 8 The cells were resuspended in 500 μL buffer to obtain a cell suspension.

[0052] (4) Use clean tweezers to mount the MS column on the MACS MultiStand sorting stand, add 500 μL of buffer to rinse the column, and then add the cell suspension in step (3).

[0053] (5) Rinse the column with 500 μL of buffer and repeat three times.

[0054] (6) Remove the column with tweezers and place it in a 5 mL centrifuge tube. Add 1 mL of buffer to the column and quickly flush the buffer into the centrifuge tube with a plunger. After slowly rotating the plunger, add 1 mL of buffer again to repeat the flushing. Centrifuge at 450 g, 4 °C, and 10 min.

[0055] (7) Discard the supernatant, resuspend the cells in an appropriate amount of PBS, and count the live cells by trypan blue staining.

[0056] 3. Cultivating cells

[0057] (1) The cells obtained in the above step were centrifuged at 800 rpm for 5 min.

[0058] (2) Discard the supernatant, resuspend the cells in culture medium according to the count and adjust the cells to an appropriate culture concentration. Transfer the cell suspension to a culture flask or dish of appropriate size, mark the detailed information of the cells on the flask or dish, and culture it in an incubator at 37°C, 5% CO2, 5% O2. 5% CO2 and 5% O2 are the percentages of gas concentration in the incubator, which will be displayed on the incubator.

[0059] (3) Depending on the cell growth status, the medium was changed every 2 to 3 days. The day of cell extraction was designated as day 0 and the maximum culture period was 28 days.

[0060] The culture system (0.001TFP) provided by the present invention is contained in a culture flask or a culture dish. As a positive control group, the culture flask or the culture dish is filled with 0.1% HSA: HSC is cultured in DMEM / F12 medium, to which 1% ITS-X, 1% P / S / G, TPO 100ng / mL, SCF 10ng / mL, and 0.1% HSA are added, and HSA is the effective component serum albumin (human serum albumin, HSA) in serum.

[0061] The culture medium contains the following components: DMEM / F12 culture medium, 1% ITS-X, 1% P / S / G, TPO 100 ng / mL, and SCF 10 ng / mL.

[0062] The 0.001% TFP group was the experimental group, in which 0.001% TFP was added to the above common ingredients without HSA.

[0063] The 0.1% HSA group was the positive control group, in which 0.1% HSA was added to the above common components without TFP.

[0064] 4. Flow cytometry detection of cell ratio

[0065] (1) Take cells at a specific time, transfer them into a flow cytometry tube, and centrifuge at 1000 rpm for 5 min.

[0066] (2) Discard the supernatant, add 2 mL of PBS, shake to mix, and centrifuge at 1000 rpm for 5 min.

[0067] (3) Discard the supernatant and add 0.625 μL of flow cytometry antibodies CD117-APC, 1.25 μL of Sca-1-PE, 20 μL of Lineage-FITC, 2.5 μL of CD34-PE / CY7, and 2.5 μL of CD135-PE / CY5 to the cells, respectively. Vortex to mix and incubate at room temperature in the dark for 30 min.

[0068] (4) Add 2 mL of PBS to the flow tube, shake to mix, and centrifuge at 1000 rpm for 5 min.

[0069] (5) Add 1 mL of PBS to the flow cytometry tube, shake to mix, place the cells on ice, and wait for loading.

[0070] (6)HSC:Lineage - Sca-1 + CD117 + ;

[0071] LT-HSC:Lineage - Sca-1 + CD117 + CD34 - CD135 - ;

[0072] ST-HSC: Lineage - Sca-1 + CD117 + CD34 + CD135 - ;

[0073] MPP: Lineage - Sca-1 + CD117 + CD34 + CD135 + .

[0074] V. Experimental Results

[0075] Figure 1 Mouse bone marrow CD117 + The results of cell expansion times in each group after 5, 7, and 9 days of in vitro expansion of cells in 0.1% HSA, 0.1% TFP, 0.01% TFP, and 0.001% TFP, respectively. It can be seen that the number of cells increased after 5, 7, and 9 days of culture in each group, and the expansion times in the 0.1% TFP, 0.01% TFP, and 0.001% TFP groups were higher than that in the 0.1% HSA group, which was becoming more and more obvious.

[0076] Figure 2 Mouse bone marrow CD117+ The proportion of each group of cells (HSC, LT-HSC, ST-HSC, MPP) after 9 days of in vitro expansion in 0.1% HSA, 0.1% TFP, 0.01% TFP and 0.001% TFP. After 9 days of culture, the proportion of HSC and LT-HSC (the main research objects and cells that play a role) in the 0.1% TFP and 0.01% TFP groups decreased compared with day 0. Therefore, although their expansion times increased, they were not considered as conditions for the subsequent culture system. The proportion of HSC and LT-HSC in the 0.1% HSA group and the 0.001% TFP group increased compared with day 0, indicating that the culture was effective and in vitro expansion did increase the proportion of HSC and LT-HSC. The proportion of HSC and LT-HSC in the 0.001% TFP group was significantly higher than that in the 0.1% HSA group, and the difference was statistically significant.

[0077] Figure 3 Mouse bone marrow CD117 + Cell morphology of each group of cells under a microscope before and after 9 days of in vitro expansion in the culture system provided by the present invention (400×). Figure 3 It can be seen that on day 0, the cells in both groups were small and round, with strong refractive index, and the cells were in good condition. After 9 days of culture, the number of cells in the 0.001% TFP group was greater than that in the 0.1% HSA group, and most cells were small in morphology, while some were large. The number of large-volume cells in the 0.1% HSA group was greater than that in the 0.001% TFP group, and large-volume cells represent cells in poor culture or differentiated cells.

Claims

1. A culture system for in vitro expansion of mouse bone marrow hematopoietic stem cells, characterized in that: The culture system uses DMEM / F12 culture medium as a basic culture medium, and is supplemented with ITS-X, P / S / G, TPO trehalose solution, SCF trehalose solution and tremella polysaccharide aqueous solution. The volume fractions of ITS-X and P / S / G in the entire culture system are both 1%, the concentration of TPO trehalose is 100 ng / mL, the concentration of SCF trehalose is 10 ng / mL, and the mass percentage concentration of tremella polysaccharide is 0.001%.

2. A culture system for in vitro expansion of mouse bone marrow hematopoietic stem cells according to claim 1, characterized in that: The concentration of the TPO trehalose solution is 10 μg / mL, the concentration of the SCF trehalose solution is 1 μg / mL, and the concentration of the Tremella polysaccharide aqueous solution is 20 mg / mL.

3. The method for preparing a culture system for in vitro expansion of mouse bone marrow hematopoietic stem cells according to claim 2, characterized in that: The following steps are involved: Step 1, dissolve 5 g Tremella polysaccharide powder in 250 mL sterile water, shake vigorously, and configure to 20 mg / mL for standby use; Step 2, first dissolve 10 μg SCF powder in 10 μL sterile water, mix well, and prepare to 1 mg / mL; then add 10 μL of the 1 mg / mL mixture to 90 μL of a 5% trehalose solution to prepare to 0.1 mg / mL; then add 100 μL of the 0.1 mg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 10 μg / mL; finally, add 100 μL of the 10 μg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 1 μg / mL; package in 500 μL tubes, store at -80°C for future use; Step 3, first dissolve 100 μg TPO powder in 200 μL sterile water, mix well, and prepare to 0.5 mg / mL; then add 200 μL of the 0.5 mg / mL mixture to 800 μL of a 5% trehalose solution to prepare to 0.1 mg / mL; finally, add 100 μL of the 0.1 mg / mL mixture to 900 μL of a 5% trehalose solution to prepare to 10 μg / mL; divide into 500 μL tubes, store at -80°C, and use; Step 4, directly package P / S / G and ITS-X into 500 μL tubes and store at -80°C for future use; Step 5, add 500 μL P / S / G, 500 μL ITSX, 500 μL of 1 μg / mL SCF in step 2, 500 μL of 10 μg / mL TPO in step 3, and 25 μL of 20 mg / mL Tremella polysaccharide in step 1 to 47.975 mL DMEM / F12 medium to prepare a serum-free Tremella polysaccharide medium with a mass percentage concentration of 0.001%.

Images