Umbilical cord blood-derived hematopoietic stem cells as well as amplification method and application thereof

Abstract

The invention discloses a cord blood-derived hematopoietic stem cell as well as an amplification method and application thereof. The method comprises the step of culturing umbilical cord blood mononuclear cells by using a culture medium containing thymus active polypeptide, stem cell growth factors and heat shock protein. According to the method for amplifying the umbilical cord blood-derived hematopoietic stem cells, the proliferation efficiency of umbilical cord blood mononuclear cells can be remarkably improved, and the amplification capacity of umbilical cord blood immune cells (NK cells) is improved by more than 5 times compared with that of a traditional method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to hematopoietic stem cells derived from umbilical cord blood and a method and application thereof for amplification. Background Art

[0002] Umbilical cord blood is rich in hematopoietic stem cells, which can rebuild the human hematopoietic system and immune system, and therefore have been widely used in hematopoietic stem cell transplantation. Hematopoietic stem cells have multidirectional differentiation potential and can differentiate into a variety of human cell types, such as blood cells, nerve cells, and bone cells. This potential makes umbilical cord blood a very valuable human biological resource, especially as a source of unrelated hematopoietic stem cells. In recent years, with the development of biomedical technology, research on the use of umbilical cord blood stem cells to treat a variety of diseases has gradually deepened, but in actual clinical applications, the application efficiency of umbilical cord blood is still low, and its biological effectiveness has not been fully exerted.

[0003] The experience of umbilical cord blood (UCB) transplantation in the past 20 years has shown that UCB has significant therapeutic effects on a variety of diseases, especially in hematological malignancies, bone marrow failure, hemoglobinopathies, and inborn errors of metabolism. However, clinically, only a few umbilical cord blood units have sufficient cell content for allogeneic hematopoietic stem cell transplantation. The main effect of UCB comes from the mononuclear cells (MNCs) therein, which include lymphocytes, monocytes, and stem cells of the hematopoietic cell lineage. Mononuclear cells can be expanded in vitro and differentiated into different types of immune cells to meet different clinical needs. However, due to individual differences in cord blood collection and time delays from collection to preparation, the activity and number of cord blood mononuclear cells are difficult to accurately control, which increases the difficulty of subsequent in vitro expansion.

[0004] Umbilical cord blood is rich in hematopoietic stem cells, which can differentiate into a variety of immune cells with clinical application value. At present, umbilical cord blood has been used for the in vitro expansion of a variety of immune cells, but traditional expansion technology has obvious shortcomings and low expansion efficiency, which makes it difficult to meet the clinical demand for high-quantity and high-activity immune cells. The number of cells obtained by traditional expansion methods is limited, and the purity is not high, which limits the application of umbilical cord blood in immune cell therapy and makes it difficult to fully realize the potential therapeutic value of umbilical cord blood.

[0005] Therefore, there is an urgent need to provide a method that can significantly improve the proliferation efficiency of umbilical cord blood mononuclear cells and the expansion capacity of umbilical cord blood immune cells. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides hematopoietic stem cells derived from umbilical cord blood and a method and application thereof for their expansion, which significantly improve the proliferation efficiency of umbilical cord blood mononuclear cells and increase the expansion capacity of umbilical cord blood immune cells (NK cells) by more than 5 times compared with traditional methods.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for expanding hematopoietic stem cells derived from umbilical cord blood, the method comprising: culturing umbilical cord blood mononuclear cells using a culture medium containing thymic active polypeptide, stem cell growth factor and heat shock protein.

[0009] The method for amplifying cord blood-derived hematopoietic stem cells of the present invention can significantly improve the proliferation efficiency of cord blood mononuclear cells, and improve the amplification capacity of cord blood immune cells (NK cells) by more than 5 times compared with traditional methods.

[0010] Thymopentin is a bioactive peptide mainly secreted by thymic cortical and medullary epithelial cells, containing a variety of small molecule peptides and active proteins.

[0011] Stem Cell Growth Factor (SCF) is a combination of highly effective proteins and enzymes developed by AgingOff Biotech in collaboration with Harvard University, University of Pennsylvania, University of Pittsburgh, Massachusetts Institute of Technology and other institutions.

[0012] Heat shock proteins (HSPs) are intracellular molecular chaperones.

[0013] Thymic polypeptides contain a variety of biologically active ingredients, which can significantly enhance the vitality and proliferation ability of umbilical cord blood mononuclear cells and promote the realization of their differentiation potential. Stem cell growth factor, as a key cytokine, can bind to the surface receptors of hematopoietic stem cells, significantly improve the survival rate, proliferation ability and differentiation efficiency of stem cells, thereby effectively enhancing their survival and immune reconstruction effects after transplantation. In addition, heat shock proteins protect cells from stress damage, maintain cell homeostasis, and improve the tolerance and activity of umbilical cord blood stem cells during transplantation. The combined use of thymic polypeptides, stem cell growth factors and heat shock proteins can significantly improve the application efficiency and therapeutic effect of umbilical cord blood hematopoietic stem cells, and promote the widespread application of umbilical cord blood resources in clinical practice.

[0014] Preferably, the concentration of thymus active polypeptide in the culture medium is 300-600 ng / mL (eg, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL).

[0015] Preferably, the concentration of the stem cell growth factor in the culture medium is 20-80 ng / mL (eg, 20 ng / mL, 30 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL).

[0016] Preferably, the concentration of heat shock protein in the culture medium is 100-500 ng / mL (eg, 100 ng / mL, 150 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL).

[0017] Preferably, the method further comprises: extracting mononuclear cells from umbilical cord blood, culturing the mononuclear cells in a serum-free medium, replenishing the serum-free medium every 2-3 days, and controlling the cell concentration to be (0.5-2)×10 6 / mL, and culture for 4-12 days.

[0018] Above (0.5-2)×10 6 You can choose 0.5×10 6 / mL, 1×10 6 / mL, 1.5×10 6 / mL, 2×10 6 Pieces / mL, etc.

[0019] Preferably, extracting mononuclear cells from umbilical cord blood comprises: centrifuging the umbilical cord blood sample at 150-450g (e.g., 150g, 250g, 450g) for 8-15min (e.g., 8min, 10min, 15min), removing the upper light yellow plasma, diluting the remaining blood with a buffer solution, adding the diluted blood dropwise onto the upper layer of lymphocyte separation solution, and centrifuging at 350-400g (e.g., 350g, 380g, 400g) for 25-35min (e.g., 25min, 30min, 35min) to obtain mononuclear cells in the middle layer; the volume ratio of the buffer solution to the remaining blood is (1-2):1.

[0020] Preferably, the serum-free culture medium contains IL-2, IL-15 and cord blood plasma.

[0021] Preferably, the concentration of IL-2 in the serum-free medium is 200-500 U / mL (e.g., 200 U / mL, 250 U / mL, 300 U / mL, 350 U / mL, 400 U / mL, 500 U / mL).

[0022] Preferably, the concentration of IL-15 in the serum-free medium is 30-80 ng / mL (e.g., 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL).

[0023] Preferably, the mass percentage of umbilical cord blood plasma in the serum-free culture medium is 5%-15% (eg, 5%, 6%, 8%, 10%, 12%, 15%).

[0024] In a second aspect, the present invention provides a hematopoietic stem cell, which is amplified by the method for amplifying hematopoietic stem cells described in the first aspect.

[0025] In a third aspect, the present invention provides the use of the hematopoietic stem cells described in the second aspect in the preparation of immune products.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention provides an optimized method for in vitro expansion of umbilical cord blood hematopoietic stem cells, which significantly improves the proliferation efficiency of umbilical cord blood hematopoietic stem cells. In addition, it can also direct the expansion and differentiation of high-number and high-activity NK cells, thereby increasing the expansion capacity of umbilical cord blood immune cells (NK cells) by more than 5 times compared with traditional methods;

[0028] (2) The method of the present invention can significantly improve the purity of umbilical cord blood immune cells (NK cells) and the stability of in vitro expansion, thereby effectively improving the yield and quality of immune cells. It also provides a more economical and efficient solution for personalized immunotherapy and regenerative medicine, and can more efficiently utilize the valuable resource of umbilical cord blood, which has important clinical application value. DETAILED DESCRIPTION

[0029] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] Thymic active polypeptide was purchased from Dosfu Biological Co., Ltd., catalog number: 69558-55-0.

[0032] Stem cell growth factor was purchased from PeproTech, catalog number: 300-19-10.

[0033] Heat shock protein was purchased from Abbexa Ltd., catalog number: abx653682.

[0034] Fetal bovine serum was purchased from Gibco, catalog number: A5669701.

[0035] Epidermal growth factor was purchased from MedChemExpress, catalog number: HY-P7109G.

[0036] Example 1

[0037] This embodiment provides a method for expanding hematopoietic stem cells.

[0038] 1. Source and collection of specimens

[0039] Collection of umbilical cord blood samples: The umbilical cord blood and thymus selected in the present invention are all from Grade II or above hospitals in Guangdong Province and the parturients have passed prenatal examinations. The time from collection to preparation of the selected umbilical cord blood is less than 24 hours.

[0040] 2. Extraction of Mononuclear Cells

[0041] 2.1. Before entering the clean room, disinfect the room with UV light for 90 minutes and the clean bench for 30 minutes. The clean bench can only be used after being turned on for 10 minutes. Wipe the table with 75% alcohol before work.

[0042] 2.2. Place the injection saline solution, waste liquid tank and centrifuge tube in the clean bench and light the alcohol lamp. Place the injection saline solution on the right side of the clean bench; place the alcohol lamp in the center of the clean bench; and place the waste liquid tank behind the alcohol lamp.

[0043] 2.3. Take three 50mL centrifuge tubes and place them on a centrifuge tube rack. Pour 30mL of normal saline into one of them for later use.

[0044] 2.4. Spray the blood bag containing concentrated umbilical cord blood with alcohol and place it in the clean bench. Take a 50mL syringe to suck the concentrated blood out of the blood bag, carefully remove the needle of the syringe, and gently transfer the concentrated blood in the syringe to two 50mL centrifuge tubes along the wall of the centrifuge tube. Pipette 10mL of saline back into the blood bag, rinse the blood bag and recover the saline into the 50mL centrifuge tube. Repeat twice until the blood bag is clean (no red blood is visible), and dilute it to 30mL with saline (if the amount of concentrated blood is large, dilute it to 35mL), and then use a 5mL pipette to gently blow and mix the diluted blood.

[0045] 2.5. Take 15mL of Ficoll lymphocyte separation solution and place it in two 50mL centrifuge tubes. Use a 5mL pipette to slowly add the diluted blood onto the Ficoll separation solution along the wall of the test tube according to the volume ratio of blood:Ficoll=1:2. Be careful to keep the two interfaces clear and do not let the blood mix into the separation solution.

[0046] 2.6. Centrifuge at 800 g for 30 min and set the centrifuge acceleration to (1,1).

[0047] 2.7. After centrifugation, gently remove the centrifuge tube. At this time, the blood has been separated into 4 layers, from top to bottom, plasma and platelet layer, buffy coat layer, separation fluid layer, polymorphonuclear leukocytes and red blood cell layer. Use a 5mL pipette to gently aspirate the top plasma layer to about 1cm close to the buffy coat layer, rotate the buffy coat layer at a constant speed and transfer it to a new 50mL centrifuge tube. Try to collect all the buffy coat layer without touching the red blood cell layer at the bottom.

[0048] 2.8. After the buffy coat layer is aspirated, add physiological saline to 45 mL and mix gently by pipetting. Centrifuge at 600 g for 10 min, with the centrifuge acceleration set to (8,8).

[0049] 2.9. After centrifugation, discard the supernatant, add physiological saline to 45 mL, gently pipette to mix, and centrifuge again to wash. Centrifuge at 300g for 10 minutes, with the centrifuge acceleration set to (8,8).

[0050] 2.10. After centrifugation, discard the supernatant to obtain the mononuclear cell pellet, and resuspend it in culture medium to obtain a mononuclear cell suspension.

[0051] 3. Induction of differentiation of mononuclear cells

[0052] One day before preparing mononuclear cells, the activator (purchased from Hangzhou Baitong Biotechnology Co., Ltd., product number: 200100) was resuspended in 20 mL of normal saline and kept at 4°C overnight.

[0053] The isolated mononuclear cells were taken at 2×10 6 / mL concentration was inoculated into a pre-coated T175 bottle containing an activator, the T175 bottle contained a basal medium, the basal medium contained 300U / mL of IL-2, 50ng / mL of IL-15, 500ng / mL of thymic active polypeptide, 50ng / mL of stem cell growth factor, 400ng / mL of heat shock protein, 10% by mass of cord blood plasma, and 10% final concentration of hydrocortisone. -6 M (5 μL was added to 50 mL system), and after inoculation, the cells were placed in a 37°C incubator for culture. This was recorded as day 0.

[0054] Closely observe the color of the culture medium and the state of the cells during the first five days of culture. If the color of the culture medium turns yellow and cell colonies appear, add fluid. If the color of the culture medium does not change much and the number of cell colonies is small, continue to culture until the culture medium turns yellow and add fluid. When the culture system is larger than 200mL, transfer the bag for culture, observe the cell growth state, and add fluid in time. The concentrations of IL-2 and IL-15 are 300U / mL and 50ng / mL, and the amount of plasma is reduced to 3%.

[0055] During Day 18-Day 21, observe the cell growth status and culture medium color, and harvest the cells to obtain the maximum cell mass when the culture medium is about to turn yellow and the nutrients are about to be consumed.

[0056] The cells were harvested, counted, and sent for flow cytometry.

[0057] Example 2

[0058] The difference from Example 1 is that the concentration of thymus active polypeptide is 300 ng / mL, the concentration of stem cell growth factor is 80 ng / mL, and the concentration of heat shock protein is 500 ng / mL.

[0059] Example 3

[0060] The difference from Example 1 is that the concentration of thymus active polypeptide is 600 ng / mL, the concentration of stem cell growth factor is 20 ng / mL, and the concentration of heat shock protein is 100 ng / mL.

[0061] Example 4

[0062] The difference from Example 1 is that the concentration of thymus active polypeptide is 100 ng / mL, the concentration of stem cell growth factor is 50 ng / mL, and the concentration of heat shock protein is 400 ng / mL.

[0063] Example 5

[0064] The difference from Example 1 is that the concentration of thymus active polypeptide is 500 ng / mL, the concentration of stem cell growth factor is 50 ng / mL, and the concentration of heat shock protein is 600 ng / mL.

[0065] Comparative Example 1

[0066] The difference from Example 1 is that the culture medium does not contain thymus active polypeptide, and its weight fraction is proportionally distributed to the weight of stem cell growth factor and heat shock protein.

[0067] Comparative Example 2

[0068] The difference from Example 1 is that the culture medium does not contain stem cell growth factor, and its weight fraction is proportionally distributed to the weight of thymic active polypeptide and heat shock protein.

[0069] Comparative Example 3

[0070] The difference from Example 1 is that the culture medium does not contain heat shock protein, and its weight fraction is proportionally distributed to the weight of thymic active polypeptide and stem cell growth factor.

[0071] Comparative Example 4

[0072] The difference from Example 1 is that the culture medium does not contain thymus active polypeptide, stem cell growth factor and heat shock protein.

[0073] Comparative Example 5

[0074] The difference from Example 1 is that the thymus active polypeptide in the culture medium is replaced by fetal bovine serum.

[0075] Comparative Example 6

[0076] The difference from Example 1 is that the stem cell growth factor in the culture medium is replaced by epidermal growth factor EGF.

[0077] Test Example 1

[0078] The NK cell proliferation numbers and flow cytometry phenotype identification results of the examples and comparative examples are shown in Table 1.

[0079] Table 1

[0080] Group <![CDATA[Number of NK cells (10 9 )]]> CD3-(%) CD3-CD16+CD56+(%) Example 1 36.76 95.22 92.81 Example 2 32.65 93.56 88.12 Example 3 33.25 91.78 86.98 Example 4 18.45 76.89 74.12 Example 5 16.98 78.26 71.12 Comparative Example 1 12.78 77.23 75.12 Comparative Example 2 15.56 82.45 72.56 Comparative Example 3 12.32 85.67 78.89 Comparative Example 4 5.21 90.12 73.67 Comparative Example 5 12.84 80.19 74.34 Comparative Example 6 14.91 84.56 69.98

[0081] It can be seen from Table 1 that the addition of thymic active polypeptide, stem cell growth factor and heat shock protein in the in vitro amplification of NK in the methods of Examples 1-3 of the present invention can significantly increase the number of in vitro amplification of NK cells, and can obtain purer NK cells. Example 4-5 illustrates that thymic active polypeptide, stem cell growth factor and heat shock protein can play a synergistic role under a specific ratio, and jointly promote the number and purity of NK in vitro amplification. Comparative Examples 1-6 illustrate that the combined application of thymic active polypeptide, stem cell growth factor and heat shock protein in the method of the present invention can significantly improve the amplification quantity and quality of umbilical cord blood immune cells, and the synergistic effect of the three substances can increase the amplification capacity of immune cells by more than 5 times compared with traditional methods.

[0082] In summary, the present invention can form a comprehensive system that promotes the activity and proliferation of mononuclear cells by jointly adding thymic active polypeptide, stem cell growth factor and heat shock protein, so that the in vitro expanded immune cells have higher biological activity, and the expansion efficiency is improved by more than 5 times compared with the traditional method, which provides a more efficient solution for immunotherapy and regenerative medicine, and has broad clinical application prospects. The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the raw materials of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for expanding hematopoietic stem cells derived from cord blood, characterized in that: The method comprises: using a culture medium containing thymus active polypeptide, stem cell growth factor and heat shock protein to culture umbilical cord blood mononuclear cells.

2. The method for expanding hematopoietic stem cells according to claim 1, characterized in that: The concentration of thymus active polypeptide in the culture medium is 300-600 ng / mL.

3. The method for expanding hematopoietic stem cells according to claim 1 or 2, characterized in that: The concentration of stem cell growth factor in the culture medium is 20-80 ng / mL.

4. The method for expanding hematopoietic stem cells according to any one of claims 1 to 3, characterized in that: The concentration of heat shock protein in the culture medium is 100-500 ng / mL.

5. The method for expanding hematopoietic stem cells according to any one of claims 1 to 4, characterized in that: The method further comprises: extracting mononuclear cells from umbilical cord blood, culturing the mononuclear cells in a serum-free medium, replenishing the serum-free medium every 2-3 days, and controlling the cell concentration to be (0.5-2)×10 6 / mL, and culture for 4-12 days.

6. The method for expanding hematopoietic stem cells according to claim 5, characterized in that: Extracting mononuclear cells from umbilical cord blood includes: centrifuging the umbilical cord blood sample at 150-450g for 8-15 minutes, removing the upper light yellow plasma, diluting the remaining blood with a buffer solution, dripping the diluted blood on the upper layer of lymphocyte separation solution, centrifuging at 350-400g for 25-35 minutes, and obtaining mononuclear cells in the middle layer; the volume ratio of the buffer solution to the remaining blood is (1-2):

1.

7. The method for expanding hematopoietic stem cells according to claim 5 or 6, characterized in that: The serum-free culture medium contains IL-2, IL-15 and cord blood plasma.

8. The method for expanding hematopoietic stem cells according to claim 7, characterized in that: The concentration of IL-2 in the serum-free medium is 200-500 U / mL; Preferably, the concentration of IL-15 in the serum-free medium is 30-80 ng / mL; Preferably, the mass percentage of cord blood plasma in the serum-free culture medium is 5%-15%.

9. A hematopoietic stem cell, characterized in that: The hematopoietic stem cells are obtained by amplifying the hematopoietic stem cells according to any one of claims 1 to 8.

10. Use of the hematopoietic stem cells according to claim 9 in the preparation of immune products.