Preparation of thymus supernatant and its application in immune cell culture

Abstract

The present invention relates to the preparation of thymic supernatant and its use in immune cell culture. Specifically, the present invention relates to a method for culturing or expanding immune cells or their progenitor cells in vitro, comprising adding an extracellular extract of thymic tissue to a cell culture medium. In another aspect, the present invention also relates to a cell culture medium comprising the extracellular extract of thymic tissue.

Description

Technical Field

[0001] The present invention relates to a method for culturing immune cells and progenitor cells thereof, and in particular to the preparation of thymus supernatant and the application thereof in culturing immune cells and progenitor cells thereof. Background Art

[0002] With the development and application of CAR-T cell therapy, T cell therapy has been widely used in recent years to treat tumors, autoimmune diseases, and other diseases. To obtain clinically applicable quantities of T cells, it is generally necessary to isolate T cells from umbilical cord blood (allogeneic) or peripheral blood (autologous) and then expand them in vitro. The key focus of in vitro T cell expansion has always been how to obtain sufficient numbers of T cells in a short period of time while maintaining their tumor-killing activity.

[0003] The method currently used to culture T cells includes culturing T cells with serum-free culture medium or culturing T cells with fetal bovine serum or human serum as culture medium additives. Among them, the advantage of using serum-free culture medium is that it is of non-animal origin, which reduces the risk of carrying animal-derived pathogens during cell amplification in vitro and reagent preparation, but the proliferation multiples of T cells obtained by culture are limited and the number is small, so they cannot be widely used for treatment, which limits the application of T cells in immune cell therapy. The advantage of using fetal bovine serum or human serum as a culture medium additive to culture T cells is that fetal bovine serum (FBS) is easy to obtain and is widely used in the culture of T cells and other types of cells, but T cells cultured with FBS are prone to adverse reactions in clinical experiments. Therefore, it has become a demand to find a composition or method that can efficiently amplify T cells and their progenitor cells in vitro.

[0004] The mammalian thymus is the site of T cell development and maturation. It is the first organ in mammals to begin aging after birth. In aging, the fibroblasts and adipocytes in the thymus multiply, while the microenvironment that supports T cell development gradually disappears. Therefore, the thymus is closely linked to the growth and development of T cells.

[0005] At present, the research on the promotion of T cell proliferation and development by thymus tissue mainly focuses on the study of thymosin, in which the thymosin is mainly a polypeptide component extracted by crushing the cells, which is an intracellular polypeptide. For example, patent CN100402085C describes that "the extraction method of thymosin solution is: take fresh or quick-frozen thymus, remove impurities and wash it; add water to the treated thymus to prepare a homogenate; repeatedly freeze and thaw the homogenate; heat the freeze-thaw solution to remove unstable impurities, and then centrifuge and filter to obtain the supernatant; ultrafiltration of the supernatant to retain components with a molecular weight of less than 10,000 Daltons to obtain a low-concentration thymosin solution." It can be seen from the above extraction method that the extraction technology of thymosin will completely crush the thymocytes and completely release the cell contents. Regarding the use of thymosin in immune cell culture, patent CN115247148B introduces thymopentin as an additive to immune cell culture medium.

[0006] However, there are currently few studies on thymic supernatant (whose main component is thymic extracellular substances) and its application. Summary of the Invention

[0007] In one aspect of the present invention, a method for culturing or expanding immune cells or progenitor cells thereof in vitro is provided, which comprises adding an extracellular extract of thymus tissue to a cell culture medium.

[0008] In a specific embodiment of the present invention, the immune cells or their progenitor cells include T cells, B cells, NK cells, NKT cells, macrophages, TIL cells, CIK cells, TCR-T cells and other tumor-killing cells, and hematopoietic stem cells.

[0009] In a specific embodiment of the present invention, the thymus tissue is obtained from a mammal, preferably a newborn mammal; optionally, the mammal comprises cattle, sheep, horses, pigs, preferably cattle.

[0010] The culture media for T cell expansion currently available on the market are generally divided into serum culture media and serum-free culture media, on which the cytokines necessary for T cell expansion (IL2, IL7, IL15, etc.) are added. The present invention first screened the commonly used culture systems on the market to determine the optimal T cell culture conditions to verify the subsequent addition of thymic tissue extracellular extracts, including the screening of basal culture media and cytokines. For example, three serum-containing culture media (1640+FBS, 1640+βME+FBS, αMEM), two serum-free culture media (X-VIVO15, ImmunoCult-XF), and the in vitro expansion rate of T cells under various cytokine combinations were compared; the optimal culture conditions were finally determined to be ImmunoCult-XF+IL7 / IL15. It should be noted that the present invention only exemplarily screened the above-mentioned culture conditions. It will be understood by those skilled in the art that the above-mentioned culture conditions are only exemplary optimal conditions. The culture conditions conventionally used for immune cells such as T cells in the prior art are applicable to the present invention, that is, the thymic tissue extracellular extract of the present invention can be applied to any conventional cell culture conditions in the prior art.

[0011] In order to create a microenvironment that is more conducive to immune cell expansion and differentiation for immune cells such as T cells and immune cell progenitor cells in in vitro amplification, the present invention adds the thymus tissue extracellular extract of the present invention (also known as newborn bovine thymus extract, Newborn bovine THYmic supernatant, THY) on the basis of the culture system currently available on the market. Compared with thymosin in the prior art or fetal bovine serum conventionally used for T cell culture in the prior art, it can obtain more numbers of immune cells (such as T cells) and immune cell progenitor cells (such as hematopoietic stem cells; HSC) in a short period of time. Specifically, it improves the in vitro amplification efficiency of T cells, maintains its typing diversity, and enhances the tumor killing ability of T cells. In addition, it is applied in the hematopoietic stem cell (immune cell progenitor cell group) culture system, and hematopoietic stem cells can be efficiently amplified.

[0012] In another aspect, the present invention provides a cell culture medium comprising an extracellular extract of thymus tissue.

[0013] The thymic tissue extracellular extract, also known as thymic supernatant, is a T cell culture additive that outperforms existing commercial expansion systems. It not only improves the in vitro expansion efficiency of T cells but also enhances their tumor-killing ability. Furthermore, its application in hematopoietic stem cell (immune cell progenitor) culture systems can efficiently expand hematopoietic stem cells.

[0014] The thymic supernatant THY is different from the thymic tissue extracts in the prior art (such as thymosin, which is mainly an intracellular polypeptide). The components of the thymic supernatant of the present invention are mainly extracellular components such as cytokines. The inventors analyzed the components of the thymic supernatant THY and compared it with the components of fetal bovine serum (FBS), which is traditionally used as a cell culture supplement. It was found that it contains a variety of common cytokines in fetal bovine serum (FBS), such as IL7, IL2, IL15, IL12, TNFα, INFγ, and GM-CSF. Moreover, under the same total protein concentration conditions, the content of these cytokines is much higher than that of fetal bovine serum.

[0015] In a specific embodiment of the present invention, an extracellular extract from bovine thymus tissue is exemplarily provided. Those skilled in the art can expect that extracellular extracts from thymus tissues of other mammals will have the same effect.

[0016] The extract of the present invention is first centrifuged at low speed (600-4000 rpm, preferably 800-3000 rpm) to remove cellular components, thereby highly enriching the extracellular active ingredients of thymocytes. Cytokines associated with immune cell expansion and activation are primarily secreted polypeptides. Therefore, compared with existing thymosin preparations, the concentration of active ingredients that support immune cell expansion is greatly increased. Secondly, the entire process is operated at low temperatures (2-10°C, preferably 4-6°C), preserving the spatially active structure of the active ingredients.

[0017] Furthermore, the present invention demonstrates through comparative experiments that thymus supernatant extract as a cell culture additive has a stronger amplification effect on immune cells than thymopentin or fetal bovine serum conventionally used for cell culture in the prior art.

[0018] In a specific embodiment of the present invention, the content of the thymus supernatant extract in the cell culture medium can be routinely adjusted as needed, such as 5%-20% or 5%-15% by volume, such as 10% by volume.

[0019] The cell culture medium further comprises cytokines known in the prior art for T cell culture, such as IL7 and IL15; preferably, the content of IL7 and IL15 is 5-15 ng / ml, such as 10 ng / ml.

[0020] Alternatively, the cell culture medium further comprises growth factors known in the prior art for maintaining stem cell proliferation, such as 5-15 ng / ml SCF, 80-120 ng / ml TPO, 80-120 ng / ml FILT3; preferably 10 ng / ml SCF, 100 ng / ml TPO, 100 ng / ml FILT3.

[0021] On the other hand, the present invention also provides a method for preparing the thymus tissue extracellular extract, which comprises adding a buffer to the thymus tissue to dissolve the thymus tissue extracellular substances in the buffer, and collecting the supernatant by centrifugation.

[0022] The centrifugation comprises firstly centrifuging at low temperature and low speed to remove cell components, and then centrifuging at low temperature and high speed to remove cell debris;

[0023] In a specific embodiment of the present invention, the buffer solution is a conventional physiological buffer solution in the prior art, such as PBS phosphate buffer solution and Hank's balanced salt solution. The amount of the buffer solution added can be conventionally selected, for example, 300 ml of buffer solution is added per 100 g of thymus tissue.

[0024] In a specific embodiment of the present invention, the low-temperature low-speed centrifugation is 600-4000 rpm, preferably 800-3000 rpm, and centrifugation at 2-10°C, preferably 2-8°C, and more preferably 4-6°C for 5-15 minutes; the low-temperature high-speed centrifugation is 6000-12000 rpm, and centrifugation at 2-10°C, preferably 2-8°C, and more preferably 4-6°C for 20-40 minutes.

[0025] In a specific embodiment of the present invention, the process further comprises removing pollutants from the supernatant; preferably, the pollutants from the supernatant are removed by membrane filtration to obtain a purified thymus tissue extracellular extract (also known as thymus supernatant).

[0026] In a specific embodiment of the present invention, the purpose of the membrane filtration treatment is to remove pollutants such as bacteria and mycoplasma. Pollutants can also be removed by ultraviolet irradiation, radiation sterilization, activated carbon adsorption and other methods.

[0027] Compared with the prior art methods for preparing thymic tissue extracts, the thymic supernatant preparation method of the present invention is simple to operate. For example, (1) the prior art methods for preparing thymic tissue extracts usually require cell disruption, that is, the cells are completely disrupted by a trypsin digestion step to release the internal contents of the cells, while the preparation method of the present invention does not require cell disruption, and retains extracellular substances to the maximum extent, which is better used to support the supplementation of nutrients during in vitro cell expansion. (2) The preparation method of the present invention reduces the high-temperature cooking step. The prior art methods for preparing thymic extracts use high-temperature treatment, which will inactivate most of the proteins. The present invention maintains low temperature throughout the process, which greatly protects the activity of effective proteins.

[0028] In the present invention, the mammal is selected from humans, mice, rats, dogs, cats, rabbits, pigs, monkeys, horses, cattle or sheep; in a preferred embodiment of the present invention, the newborn mammal refers to a newborn calf, and the newborn calf refers to a cattle within 30 days of birth, preferably within 24 hours, preferably within 14 hours of birth and without food.

[0029] In another aspect of the present invention, provided are immune cells or progenitor cells thereof obtained by the above method, and preparations containing the immune cells or progenitor cells thereof.

[0030] In a specific embodiment of the present invention, the preparation further comprises a pharmaceutically acceptable excipient.

[0031] In a specific embodiment of the present invention, the pharmaceutically acceptable excipient refers to a diluent, adjuvant or vehicle used together with the preparation of the present invention or the cell, including saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea etc. In addition, adjuvants, stabilizers, thickeners, lubricants and coloring agents can be used. In one embodiment, when applied to an animal, the pharmaceutically acceptable excipient is sterile. When administered intravenously, water is a preferred excipient. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid excipients, particularly for injection solutions.

[0032] In another aspect of the present invention, there is provided a use of immune cells or progenitor cells thereof or the preparation in preparing a medicament for treating tumors; in a specific embodiment of the present invention, the tumor includes a solid tumor or a non-solid tumor.

[0033] In a specific embodiment of the invention, the tumor can be selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or inside the eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, kidney cancer, soft tissue sarcoma, urethral cancer, bladder cancer, renal cancer, lung cancer, non-small cell lung cancer, thymoma, prostate cancer, mesothelioma, adrenocortical cancer, lymphoma, for example Hodgkin's disease, non-Hodgkin's disease, gastric cancer, leukemias such as ALL, CLL, AML, urothelial leukemia and multiple myeloma.

[0034] In one embodiment, the tumor is a solid tumor. Examples of solid tumors that can be treated accordingly include breast cancer, lung cancer, colorectal cancer, pancreatic cancer, gliomas, and lymphomas. Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS, neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and brain metastases, melanomas, gastrointestinal and renal cancers and sarcomas, rhabdomyosarcomas, glioblastomas, preferably glioblastoma multiforme and leiomyosarcomas. Examples of vascularized skin cancers for which the antagonists of the present invention are effective include squamous cell carcinomas, basal cell carcinomas, and skin cancers that can be treated by inhibiting the growth of malignant keratinocytes, such as human malignant keratinocytes.

[0035] In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma, and lymphoma.

[0036] Specifically, the present invention provides the following technical solutions:

[0037] 1. A method for culturing or expanding immune cells or progenitor cells thereof in vitro, comprising adding an extracellular extract of thymus tissue to a cell culture medium.

[0038] 2. The method according to item 1, wherein the immune cells or their progenitor cells include T cells, B cells, NK cells, NKT cells, macrophages, TIL cells, CIK cells, TCR-T cells and other tumor-killing cells, and hematopoietic stem cells.

[0039] 3. The method according to item 1 or 2, wherein the thymus tissue is obtained from a mammal, preferably a newborn mammal; optionally, the mammal comprises cattle, sheep, horses, pigs, preferably cattle.

[0040] 4. The method according to item 1 or 2, wherein the cell culture medium further comprises IL7 and IL15; preferably, the concentrations of IL7 and IL15 are 5-15 ng / ml, more preferably 8-12 ng / ml, respectively;

[0041] Preferably, the basal medium of the cell culture medium is 1640 medium, 1640+β-ME medium, αMEM medium, Lonza X-vivo15 medium or ImmunoCult-XF medium; preferably, ImmunoCult-XF medium.

[0042] 5. The method according to item 1 or 2, wherein the cell culture medium further comprises 5-15 ng / ml SCF, 80-120 ng / ml TPO, and 80-120 ng / ml FILT3; preferably, the basal medium of the cell culture medium is IMDM medium.

[0043] 6. An immune cell or a progenitor cell thereof obtained by the method according to any one of items 1 to 5.

[0044] 7. A cell culture medium comprising an extracellular extract of thymus tissue; preferably, the content of the extracellular extract of thymus tissue is 5%-20% by volume.

[0045] 8. The cell culture medium according to item 7, further comprising IL7 and IL15; preferably, the concentrations of IL7 and IL15 are 5-15 ng / ml, more preferably 8-12 ng / ml, respectively;

[0046] Preferably, the basal medium of the cell culture medium is 1640 medium, 1640+β-ME medium, αMEM medium, Lonza X-vivo15 medium or ImmunoCult-XF medium; preferably, ImmunoCult-XF medium.

[0047] 9. The cell culture medium according to item 7, further comprising 5-15 ng / ml SCF, 80-120 ng / ml TPO, and 80-120 ng / ml FILT3; preferably, the basal medium of the cell culture medium is IMDM medium.

[0048] 10. Use of an extracellular extract of thymus tissue or the cell culture medium according to any one of items 7 to 9 for culturing or expanding immune cells or progenitor cells thereof in vitro.

[0049] 11. A method for preparing an extracellular extract of thymus tissue, comprising adding a buffer to the thymus tissue to dissolve the extracellular substances of the thymus tissue in the buffer, and collecting the supernatant by centrifugation.

[0050] 12. The preparation method according to item 11, wherein the centrifugation comprises first low-temperature low-speed centrifugation and then low-temperature high-speed centrifugation;

[0051] Optionally, the low-temperature low-speed centrifugation is 600-4000 rpm, preferably 800-3000 rpm, at 2-10°C, preferably 2-8°C, more preferably 4-6°C, for 5-15 minutes; optionally, the low-temperature high-speed centrifugation is 6000-12000 rpm, at 2-10°C, preferably 2-8°C, more preferably 4-6°C, for 20-40 minutes.

[0052] 13. A preparation comprising the immune cells or progenitor cells thereof according to item 6; optionally, the preparation further comprises a pharmaceutically acceptable excipient.

[0053] 14. Use of the immune cells or progenitor cells thereof described in Item 6 or the preparation described in Item 13 in the preparation of a medicament for treating a tumor; optionally, the tumor comprises a solid tumor or a non-solid tumor.

[0054] Technical effects:

[0055] 1. The present invention provides a thymus supernatant of newborn animals and a method for separating and purifying the same: the present invention can extract a supernatant containing multiple active ingredients from the thymus of newborn mammals, which retains the macromolecular proteins and exosome vesicles naturally present in the thymus of young animals, as well as other non-protein active ingredients, and removes other impurities, bacteria, and mycoplasma, thereby meeting the requirements of cell culture.

[0056] 2. Compared with the existing technology, the present invention uses THY as an additive for T cell culture. T cells can not only be rapidly expanded in a short period of time, but the expanded T cells also have stronger tumor killing ability.

[0057] 3. The present invention uses animal-derived thymus supernatant to culture T cells, which is more readily available and suitable for large-scale production. Given that the T cells amplified by the present invention have been widely used in the treatment of various tumors, the THY prepared by the present invention has good application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 A flow chart for the preparation of newborn calf thymus supernatant is shown;

[0059] Figure 2 The results of quantitative analysis of cytokines contained in the supernatant of newborn calf thymus THY are shown;

[0060] Figure 3 The effect of calf thymus supernatant (THY) on the morphology of T cell expansion in vitro was shown;

[0061] Figure 4 The effect of calf thymus supernatant THY on the cell multiplier of T cell expansion in vitro was shown;

[0062] Figure 5 The effect of newborn calf thymus supernatant THY on T cell apoptosis was shown;

[0063] Figure 6 The effect of newborn calf thymus supernatant THY on T cell expansion and typing in vitro was shown;

[0064] Figure 7The results show that T cells cultured with THY supernatant of newborn calf thymus can kill lung cancer cells A549.

[0065] Figure 8 The results show that T cells cultured with calf thymus supernatant THY can kill the hematologic malignancy cell line NALM6.

[0066] Figure 9 It was shown that newborn calf thymus supernatant THY promoted T cell CD107a degranulation;

[0067] Figure 10 It was shown that newborn calf thymus supernatant THY promotes T cell perforin secretion;

[0068] Figure 11 The figure shows that T cells expanded from calf thymus supernatant (Thy) prolong mouse survival. Figure A shows the animal experiment model. Figure B shows the survival curves of mice in each group after Thy-expanded peripheral blood-derived T cells were injected into a tumor mouse model. Figure C shows the survival curves of mice in each group after Thy-expanded umbilical cord blood-derived T cells were injected into a tumor mouse model.

[0069] Figure 12 The images show that T cells expanded from calf thymus supernatant (THY) slow down tumor progression. A shows images of residual tumor size in mice on days 7, 14, 21, and 28 after THY-expanded peripheral blood-derived T cells (PBMCT) were injected into a tumor mouse model. B shows images of residual tumor size in mice on days 7, 18, 28, and 39 after THY-expanded umbilical cord blood-derived T cells (UBMCT) were injected into a tumor mouse model.

[0070] Figure 13 The results show the effect of newborn calf thymus supernatant THY on the number of hematopoietic stem cells expanded in vitro;

[0071] Figure 14 The results show the effect of calf thymus supernatant THY on the colony-forming ability of hematopoietic stem cells.

[0072] Figure 15 It shows that the supernatant of newborn calf thymus THY has a stronger expansion effect on T cells than thymopentin, thymosin α, and thymosin β4 in the existing technology.

[0073] Figure 16 The results of screening for T cell in vitro expansion media are shown. A shows the expansion of PBMC T cells after 7 days of culture in three different serum-containing media; B shows the expansion of T cells after 7 days of culture in αMEM medium compared to two other serum-free media; and C shows a comparison of T cell expansion rates in response to various cytokine combinations. DETAILED DESCRIPTION

[0074] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0075] The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0076] Example 1: A method for extracting calf thymus supernatant. The method comprises the following steps:

[0077] 1. Buffer Preparation: Use a sterile physiological buffer solution, such as PBS (phosphate buffered saline) or Hank's balanced salt solution. Preferably, add 200 IU / mL heparin sodium to the buffer solution for anticoagulation, and a 1 / 100 volume ratio of protease inhibitors to inhibit protein degradation. Stir the buffer solution thoroughly and store on ice.

[0078] 2. Obtaining thymus tissue from newborn calves: Take thymus tissue from newborn calves (including cattle, dairy cows, and Angus cattle, regardless of gender), rinse it with saline, and place it in a sterile tray.

[0079] 3. Use the surgical scissors after high temperature disinfection to remove and discard the fascia and unnecessary fat on the surface of the thymus tissue, and cut the remainder into small pieces of about 2mm in size. Add the pre-cooled buffer prepared so that the thymus fluid contents are dissolved and released in the buffer. Preferably, buffer is added according to a certain volume ratio, for example: every 100g of thymus tissue adds 300ml of pre-cooled buffer. After fully stirring, soak it on ice for 10-30 minutes to release the thymus contents in the buffer.

[0080] 4. Transfer the buffer containing the thymus contents into sterile 50ml centrifuge tubes and centrifuge to remove the cell pellet. Specifically, centrifuge at 2000 rpm and 4°C for 10 minutes and collect the supernatant. Centrifuge the supernatant further at 10,000 rpm and 4°C for 30 minutes. Transfer the supernatant to a new sterile container.

[0081] 5. Under sterile conditions, the resulting mixture solution is filtered through a membrane filter to remove contaminants such as bacteria and mycoplasma. Such sterilization methods include, but are not limited to, ultraviolet irradiation, radiation sterilization, activated carbon adsorption, and membrane filtration. Preferably, the mixture solution is filtered once through a 0.45 μm filter membrane and then once through a 0.22 μm filter membrane. The resulting supernatant is then filtered three times through a 0.1 μm filter membrane. The extract is then stored in a separate container at -80°C.

[0082] 6. The supernatant obtained above is thymus supernatant (THY), which can be used for subsequent cell culture and other applications. Figure 1 shown.

[0083] Furthermore, the present invention also changes the centrifugation conditions, which can also obtain thymic supernatant (THY), and the thymic supernatant (THY) can still achieve the technical effects of the present invention. For example, first centrifuge at 600 rpm and 2°C for 15 minutes to collect the supernatant; then further centrifuge the obtained supernatant at 12,000 rpm and 2°C for 20 minutes to obtain thymic supernatant THY; or first centrifuge at 4,000 rpm and 10°C for 5 minutes to collect the supernatant; then further centrifuge the obtained supernatant at 6,000 rpm and 2°C for 40 minutes to obtain thymic supernatant THY.

[0084] Example 2: Quantitative analysis results of cytokines contained in calf thymus supernatant THY.

[0085] The calf thymus supernatant THY obtained in Example 1 was mixed with fetal bovine serum FBS (#10091148; Gibco TM ) Cytokine determination was performed by enzyme-linked immunosorbent assay. Figure 2 As shown, several common cytokines found in fetal bovine serum (FBS) were detected in the calf thymus supernatant (THY), including IL7, IL2, IL15, IL12, TNFα, INFγ, and GM-CSF. Furthermore, at the same total protein concentration, the levels of these cytokines were significantly higher than those in FBS. Given that these cytokines play an important role in regulating cell proliferation of immune cells (such as T cells, NK cells, DCs, and macrophages) and maintaining the characteristics of hematopoietic stem cells, these experimental results suggest that the calf thymus supernatant (THY) prepared by the above method contains a large number of active ingredients.

[0086] Example 3: Effect of Calf Thymus Supernatant (THY) on the Morphology of T Cells Expanded in Vitro

[0087] The T cells used in this invention come from two sources: one is obtained from the peripheral blood of healthy volunteers, named PBMCT; the other is obtained from the umbilical cord blood of healthy pregnant volunteers, named UBMCT. Both T cell extraction and preparation methods use CD3 microbeads (#130-097-043; Miltenyi Biotec) and are extracted according to the reagent instructions. After enrichment, the T cells are inoculated into T25 culture flasks, with a starting cell number of 1×10 per flask. 5Compare the growth of T cells in the experimental group and the control group. Observe the cell morphology and size under a microscope to reflect the cell activity.

[0088] Before the experiment, we conducted preliminary experiments on T cell expansion medium currently available on the market to determine the optimal culture conditions. T cell expansion medium is generally a basal medium supplemented with growth factors necessary for expansion. Figure 16 A shows the expansion of PBMC T cells after 7 days of culture in three different serum-containing media (1640 medium, 1640 + β-ME medium, and αMEM medium). The results show that T cells expand faster in αMEM-based medium. Figure 16 Figure B shows the T cell expansion after 7 days of culture in αMEM medium and two other serum-free media (Lonza X-vivo15 medium and ImmunoCult-XF medium). The results demonstrate that the serum-free medium ImmunoCult-XF allows for faster T cell expansion. Figure 16 Figure C compares T cell expansion rates under various cytokine combinations (IL2, IL12, IL2 / IL12, IL7 / IL12, IL15 / IL12, and IL7 / IL15; concentration: 10 ng / ml). The results demonstrate that the IL7 / IL15 combination has the fastest T cell expansion rate. Therefore, in the following implementation experiment, ImmunoCult-XF medium was selected as the basal medium, and the cytokine condition was 10 ng / ml IL7 / IL15.

[0089] In this example, the T cell culture steps included: the experimental group "THY" culture medium composition was ImmunoCult-XF medium (#10981; STEMCELL Technology), 10 ng / mL IL7 / IL15, and 10% by volume of the above-mentioned newborn calf thymus supernatant THY. Dynabeads were added to this culture medium every three days. TM Human T-Activator CD3 / CD28 magnetic beads (#11132D; Thermo Fisher) were used as T cell activators. The control group (PBS) culture medium consisted of ImmunoCult-XF medium (#10981; STEMCELL Technology), 10 ng / mL IL7 / IL15, and 10% PBS buffer by volume. Dynabeads were added to this medium every three days. TMHuman T-Activator CD3 / CD28 magnetic beads (#11132D; Thermo Fisher) were used as T cell activators. A Zeiss Axio Observer 7 inverted microscope was used to photograph and record cell morphology and number on Day 0, Day 1, Day 3, Day 5, Day 7, Day 9, Day 11, and Day 14 of culture. The results of microscopic observation on Day 7 ( Figure 3 ) It can be seen that both the control and experimental groups exhibited multiple densely growing cell clusters surrounded by scattered single cells. Normal T cell clustering is crucial for in vitro expansion and maintenance of T cell activity. Therefore, THY-treated T cells maintained the basic morphology and clustered colony characteristics of PBMCT cells.

[0090] Example 4: Effect of Calf Thymus Supernatant THY on the Cell Multiplier of T Cells in Vitro

[0091] In this example, the effect of THY on the cell multiplier of T cell expansion in vitro was studied. The control group "PBS" medium composition was ImmunoCult-XF medium (#10981; STEMCELL Technology), 10ng / mL IL7 / IL15, 10% PBS buffer by volume; Dynabeads were added to this medium every three days. TM Human T-Activator CD3 / CD28 magnetic beads (#11132D; Thermo Fisher) were used as T cell activators. The experimental group "THY" culture medium consisted of ImmunoCult-XF medium (#10981; STEMCELL Technology), 10 ng / mL IL7 / IL15, and a 10% volume ratio of the above-mentioned newborn calf thymus supernatant THY. Dynabeads were added to this culture medium every three days. TM Human T-Activator CD3 / CD28 magnetic beads (#11132D; Thermo Fisher) were used as a T cell activator. The total cell count was recorded using a CountStar cell analyzer on Day 0, Day 1, Day 3, Day 5, Day 7, Day 9, Day 11, and Day 14 after culture. The cell proliferation fold was analyzed and plotted. ** indicates a p-value less than 0.01.

[0092] Figure 4It can be seen that under the control culture conditions, after 14 days of in vitro expansion, peripheral blood-derived T cells can expand 37-fold, and umbilical cord blood-derived T cells can expand 151-fold within the same time period. Under THY culture conditions, peripheral blood-derived T cells expand 89-fold within two weeks, and umbilical cord blood-derived T cells expand 558-fold. The results show that the addition of the thymus supernatant of the present invention can effectively increase the efficiency of T cell in vitro expansion compared to the control group. Therefore, the addition of the thymus extract of newborn animals described in the present invention can greatly improve the efficiency of T cell in vitro expansion.

[0093] Example 5: Effect of Calf Thymus Supernatant THY on T Cell Apoptosis

[0094] In this embodiment, the effect of THY on T cell apoptosis was studied. Among them, the use of 7AAD and Annexin V-PE channels to detect apoptosis in cell amplification in vitro is a common method in the prior art. During flow cytometry analysis, normal cells showed double negativity in both 7AAD and Annexin V channels, while early apoptotic cells showed single positivity for Annexin V and negative for 7-AAD, and late apoptotic cells were indicated by double positivity for 7AAD and Annexin V. After culturing the experimental and control groups (wherein, the culture conditions of the experimental and control groups refer to Examples 3 and 4 above) for 7 days, the cells were collected, centrifuged at 300g for 5 minutes, and the supernatant was removed. The cells were resuspended in complete culture medium and counted, and 5X10 5 The cells were counted and resuspended in 100 μl. According to conventional flow cytometry operation methods, a blank control group was set up to adjust the instrument voltage, an antibody single staining group was used to adjust the compensation, and an FMO control group was used to determine the accuracy of the gate and the experimental group. First, 5 μl of blocking Fc receptor (#130-059-901; Miltenyi Biotec) was added to reduce nonspecific binding during the staining process. Then, the corresponding antibody (#559763; BD Pharmingen) was added and mixed well. After incubation in a 4-degree refrigerator away from light for 30 minutes, 500 μl of staining buffer was added to resuspend the cells, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 200 μl of staining buffer, detected using a Beckman CytoFLEX S flow cytometer, and analyzed using FlowJO software.

[0095] from Figure 5 Flow cytometry analysis revealed that the premature apoptosis rate of T cells cultured with calf thymus supernatant (THY) decreased from 13.6% in the control group to 5.38%. The number of late apoptotic cells also decreased from 0.51% in the control group to 0.18%. Therefore, calf thymus supernatant (THY) significantly reduces the apoptosis rate during T cell culture.

[0096] Example 6: Effect of Calf Thymus Supernatant THY on T Cell Expansion and Phenotyping in Vitro

[0097] T cells are a heterogeneous cell population. According to their linear differentiation relationship, T cells can be divided into naive To the stem cell memory (Stem Cell Memory, SCM), central memory (Central Memory, CM), effector memory (Effector Memory, EM), and finally to the terminal effector (Terminal Effector, TE, or also known as EMRA) stage. Among them, central memory T cells (TCM) are differentiated from immature T cells after encountering specific antigens and receiving appropriate co-stimulatory signals. Generally speaking, the proportion of TCM is relatively small in childhood, but with the increase of age and the atrophy of the thymus, it maintains an increasing trend and maintains a relatively stable proportion. Effector memory T cells (TEM) are also differentiated from immature T cells. However, compared with central memory T cells, effector memory T cells have stronger immediate effect functions and can quickly produce cytokines and cytotoxic effects, but have lower proliferation and lymphatic homing potential. In this example, the experimental group and the control group (wherein, the culture conditions of the experimental group and the control group refer to the above examples 3 and 4) were cultured for 7 days, and then the cells were collected, centrifuged at 300g for 5min, and the supernatant was removed. The cells were resuspended in complete culture medium and counted, and 5X10 5 The cells were counted and resuspended to 100ul. According to conventional flow cytometry operating procedures, a blank control group was set up to adjust the instrument voltage, an antibody single staining group was used to adjust the compensation, and an FMO control group was used to determine the accuracy of the gate and the experimental group. First, 5μl of blocking Fc receptor (#130-059-901; Miltenyi Biotec) was added to reduce nonspecific binding during the staining process. Then, the corresponding antibody was added and mixed, and the mixture was placed in a 4-degree refrigerator in the dark and incubated for 30 minutes. At the end of the incubation, 500ul of staining buffer was added to resuspend the cells, and the cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 200ul of staining buffer, detected using a Beckman CytoFLEX S flow cytometer, and finally analyzed using FlowJO software. The antibodies used are: CD4-PerCP (#300528; Biolegend), CD8-BV510 (#344732; Biolegend), CCR7-BV421 (#353208; Biolegend) CD45RA-PECY7 (#560675; BD Pharmingen).

[0098] from Figure 6It can be seen that THY did not change the proportion of CD4+ T cells and CD8+ T cells during in vitro expansion. At the same time, it also maintained the differentiation diversity of TN (T naive), TE (T effector), TEM (effector memory T cells, TEM), and TCM (central memory T cells, TCM); for example, compared with the control group, the diversity of TEM (effector memory T cells, TEM), TCM (central memory T cells, TCM), and TN (T naive) types was maintained.

[0099] Example 7: Cytotoxicity of T cells cultured with calf thymus supernatant (THY) against lung cancer cells A549

[0100] T cells are the immune system's primary force in killing tumor cells and are a key area of ​​research into immunotherapy heterogeneity. T cell immune responses are carried out through TCRs. When the body is stimulated by an antigen, the TCR interacts with the MHC-presented antigen complex, generating effector lymphocytes that can penetrate tissues and eliminate the target antigen, as well as memory cells that can persist and provide long-term protection in the event of a repeat antigen attack. Each T cell possesses a unique TCR molecule on its surface.

[0101] In order to verify the killing effect of T cells amplified by THY on tumors, this example selected lung cancer A549 cells (#CL-0016; Procell / Punosai) with mCherry fluorescent label as tumor killing targets. A549 cells (5×10 5 cells) and T cells (5×10 5 Cells / group were co-cultured at a 1:1 effector-target ratio (see Examples 3 and 4 above for the culture conditions of the experimental and control groups). After 24 hours, the mixed cells were harvested and labeled with a CD3 antibody (#557832; BD Pharmingen) in the Apccy7 channel for T cells, and with an mCherry channel for A549 tumor cells. Flow cytometer detection was used, and analysis was performed using FlowJO software.

[0102] The results are as follows Figure 7 As shown in the left figure, after 24 hours of in vitro co-culture, the A549 cells in the control group still had a 29% survival rate compared to the initial cell count, while after the THY-treated group T cells were co-cultured with A549 cells, the survival rate of tumor cells was only 7.14%. The experiment was repeated three times and statistically analyzed. The results are as follows Figure 7As shown in the right figure, THY-cultured T cells have a significant killing effect on tumor cells A549 compared with the control group.

[0103] Example 8: Cytotoxicity of T cells cultured with calf thymus supernatant THY against the hematologic malignancy cell line NALM6

[0104] In order to further verify the killing effect of T cells amplified by THY on tumors, this example selected another human B lymphocyte leukemia tumor cell line NALM6 (#CL-0701; Procell / Punosai) with mCherry fluorescent label as the killing target. Similarly, NALM6 cells (5×10 5 cells) and T cells (5×10 5 Cells / group were co-cultured (effector-target ratio 1:1). After 24 hours, the mixed cells were harvested and labeled with a CD3 antibody (#557832; BD Pharmingen) in the Apccy7 channel for T cells, and NALM6 tumor cells were labeled with the mCherry channel. Flow cytometry was performed using a Beckman CytoFLEX S flow cytometer and analyzed using FlowJO software.

[0105] Flow cytometry results ( Figure 8 Left) It can be observed that after T cells cultured with calf thymus supernatant THY were co-cultured with NALM6, the survival rate of NALM6 cells was lower (18.52% survival rate in the experimental group vs. 73.4% survival rate in the control group). The experiment was repeated three times and statistically analyzed. The results are as follows Figure 8 As shown in the right figure, T cells cultured with newborn calf thymus supernatant THY have a stronger killing effect on tumor cells NALM6 than the control group.

[0106] Example 9: Calf thymus supernatant THY promotes T cell CD107a degranulation

[0107] Perforin and granzymes are two important molecules involved in the killing of tumor cells by cytotoxic T cells. After recognizing tumor cells, cytotoxic T cells release perforin, which forms pores on the target cell membrane, allowing granzymes to enter the cell, thereby triggering tumor cell apoptosis. When cytotoxic T cells kill target cells, toxic granules will reach the cell membrane and fuse with the cell membrane. At this time, lysosomal-associated membrane protein-1 (CD107a molecule) is transported to the cell membrane surface, causing the release of granule contents, ultimately leading to the death of the target cell. Therefore, the CD107a molecule is a marker of cytotoxic T cell degranulation, and its expression level is significantly correlated with the killing activity of T cells.

[0108] This example uses NALM6 cells (#CL-0701; Procell / Punosai), and the culture medium is supplemented with 10% FBS (#10091148; Gibco TM ) in DMEM medium. Set up T cell killing experiment: adjust the concentration of NALM6 cells to 10 6 cells / mL, and the T cell concentration obtained after 14 days of in vitro expansion was adjusted to 5×10 6 Cells / mL, effector-target ratio (E:T) of 5:1. The experiment was divided into four groups: basal degranulation level group (100μl PBMC-T + 100μl DMEM culture medium), control group (100μl PBS-T + 100ul NALM6 cells), experimental group (100μl THY-T + 100μl NALM6 cells), positive control group (100μl PBMC-T + 100ul DMEM culture medium containing 100ng / mL PMA and 2μg / mL ionomycin). Among them, the culture conditions of THY-T and PBS-T refer to the above-mentioned Examples 3 and 4, and the PBMC-T culture conditions are the same as the PBS-T cell culture conditions. Among them, PMA / ionomycin are the two most common cell stimulants, which serve as positive controls to stimulate the activation of T cells. According to the settings of the four groups, they were added to a 96-well U-shaped plate, so that each well had a total volume of 200μl. Next, 10 μL of anti-human CD107a antibody (#560664; BD Pharmingen) was added to each well and incubated at 37°C and 5% CO2 for 4 hours. After incubation for 1 hour, 2 μL of monensin was added to block the endocytosis of CD107a molecules expressed on the cell surface and mixed by pipetting. After a total incubation of 4 hours, the liquid in each well was transferred to a 5 mL flow tube, the cells were washed twice with 1× PBS (centrifuged at 400 × g for 5 minutes at room temperature), the supernatant was removed, and the cell pellet was resuspended in 300 μL of 1× PBS. The cells were kept on ice and protected from light until collected on the machine.

[0109] like Figure 9 As shown in the results, the CD107a degranulation rate of T cells in THY culture conditions was 18.9%, which was much higher than the CD107a degranulation level of the control group (5.08%). Therefore, T cells cultured with THY supernatant of newborn calf thymus have stronger cytotoxic activity.

[0110] Example 10: Calf Thymus Supernatant THY Promotes T Cell Perforin Secretion

[0111] After the expansion of cytotoxic T cells, their effective killing of target cells depends on the contact between the T cells and target cells, and the secretion of perforin and granzyme B. Perforin can bind to the cell membrane and create holes in the cell membrane, thereby destroying the outer membrane of the target cell, allowing the release of granzyme B into the target cell, which then undergoes an enzyme-linked reaction and triggers apoptosis of the target cell.

[0112] In this example, the concentration of NALM6 cells was adjusted to 10 per ml. 6 The T cell concentration of the experimental group and the control group (wherein, the culture conditions of the experimental group and the control group refer to the above Examples 3 and 4) was adjusted to 5×10 cells per ml. 6 Cells were plated to achieve an effector-target ratio (E:T) of 5:1. Two sets of cells were added to 96-well U-shaped plates, with 10 ng / mL of IL7 / IL15 added to both the control and experimental groups. The 96-well plates were incubated overnight in a cell culture incubator. After incubation, the cell supernatant was collected and centrifuged at 300 g for 5 minutes. The supernatant was then aspirated. Perforin levels were determined using the Human Perforin ELISA Kit (PRF1) (#ab46046; ABCAM) according to the kit instructions, and statistical analysis was performed and plotted.

[0113] Figure 10 The results showed that after co-culturing T cells from two sources of THY culture (CD3+T cells isolated from umbilical cord blood and CD3+T cells from peripheral blood, i.e., UBMCT (UBT) and PBMCT (PBT)) with tumor cells, both experimental groups could detect higher levels of perforin secretion in the culture medium supernatant (>1500pg / ml) compared with the control group.

[0114] Example 11: T cells expanded from calf thymus supernatant THY prolong the survival of mice

[0115] In order to verify whether T cells expanded from calf thymus supernatant THY and injected into mice have the same tumor killing effect, Figure 11 As shown in A, this embodiment uses two batches of animal models:

[0116] A batch of mice was used 3X10 5 NALM6-luc tracer tumor cells (#CL-0701; Procell) were injected into immunodeficient mice via the tail vein. After 48 hours, immunodeficient mice with hematologic tumor models were obtained. This group of mice consisted of 12 mice, which were used for subsequent PBMCT (T cells isolated from peripheral blood) cell therapy experiments. The 12 mice obtained from the treatment were divided into three groups: (1) a non-treatment group that was not treated with any cell therapy and was only injected with tumor cells; Figure 11As shown in yellow in the broken line graph B; (2) The therapeutic effect of PBMCT cells obtained under PBS culture conditions, as shown in Figure 11 As shown by the green line in the broken line graph B; (3) The therapeutic effect of PBMCT cells obtained under THY culture conditions, such as Figure 11 B is shown by the red line in the line chart.

[0117] Another group of mice were treated with 5X10 5 The number of tumor cells / mouse NALM6-luc tracer tumor cells were injected into immunodeficient mice through the tail vein. After 48 hours, immunodeficient mice with hematologic tumor models were obtained. The number of mice in this group was 7 and was used for subsequent UBMCT (T cells separated from umbilical cord blood) cell therapy experiments. The 7 mice obtained from the treatment were divided into three groups: (1) a non-treatment group that was not treated with any cell therapy and was only injected with tumor cells, such as Figure 11 (2) The therapeutic effect of UBMCT cells obtained by PBS culture conditions, as shown in yellow in the broken line graph C. Figure 11 As shown by the green line in the line graph C. (3) The therapeutic effect of UBMCT cells obtained under THY culture conditions, as shown in Figure 11 C is shown by the red line in the line chart.

[0118] The immunodeficient mice used in this example were huHSC-NCG mice from Guangdong Yaokang Co., Ltd. After successful modeling, the survival and mortality rates of the mice were recorded on Day 0, Day 7, Day 14, Day 21, Day 28, Day 35, and Day 42, and survival curves were plotted.

[0119] like Figure 11 As shown, the PBMCT group of mice derived from peripheral blood cultured with calf thymus supernatant THY significantly prolonged the survival period of mice compared with the PBS control group (the PBMCT and UBMCT derived from peripheral blood cultured in the THY and PBS control groups are shown in Example 4) (11B): all mice in the untreated group died within four weeks, the longest growth period of mice in the PBS-treated group was 30 days, and the longest growth period of mice in the THY group was extended to 38 days after cell therapy. The UBMCT group of mice derived from umbilical cord blood cultured with calf thymus supernatant THY also showed a longer survival period than the PBS control group (11C): all mice in the untreated group died within three weeks, the longest growth period of mice in the PBS-treated group was 46 days, and the longest growth period of mice in the THY group was extended to 52 days after cell therapy.

[0120] Example 12: T cells expanded from calf thymus supernatant (THY) slow down tumor progression

[0121] This example studies the effect of THY-expanded T cells on the progression of tumors. Two animal models were used: one group of mice was treated with 3×10 5 Number of tumor cells / mouse NALM6-luc tracer tumor cells were injected into immunodeficient mice through the tail vein. After 48 hours, immunodeficient mice with hematologic tumor model were obtained. This group of mice had 12 mice and was used for subsequent PBMCT cell therapy experiments. Another group of mice was treated with 5X10 5 Tumor cells (number / mouse) were injected with NALM6-luc-tracing tumor cells into immunodeficient mice via the tail vein. Forty-eight hours later, immunodeficient mice with a hematologic tumor model were obtained. This group of seven mice was used for subsequent UBMCT cell therapy experiments. The experimental procedure in this example was identical to that in Example 11. The immunodeficient mice used in this example were huHSC-NCG mice from Guangdong Yaokang Company. After successful modeling, images were taken and recorded using the multimodal animal in vivo imaging system AniView100Pro on Days 7, 14, 21, 28, and 39.

[0122] like Figure 12 As shown, the AniView100 multi-mode animal in vivo imaging system was used to record the tracer tumor cells NALM6-Luc injected into the tail vein of mice. The intensity of the tumor signal was used to represent the size of the tumor. It can be seen that in the newborn calf thymus supernatant THY experimental group, both peripheral blood-derived T cells ( Figure 12 A) or T cells from umbilical cord blood ( Figure 12 B) The time it took to capture tumor cell signals was later than that of the control group, and at the same time point, the tumor signals in the control group mice were stronger than those in the THY group. This suggests that T cells treated with calf thymus supernatant in the THY experimental group have a certain effect on delaying tumor progression.

[0123] Example 13: Effect of Calf Thymus Supernatant (THY) on the Number of Hematopoietic Stem Cells Expanded in Vitro

[0124] Hematopoietic stem cells are the progenitors of various immune cells and possess the ability to self-renew, multidirectionally differentiate, and home to their home ancestry. Hematopoietic stem cell transplantation is used to treat diseases such as leukemia and immune system abnormalities.

[0125] In this example, the effect of the above-mentioned newborn calf thymus supernatant THY on the in vitro expansion of human hematopoietic stem cells HSC (CD34+ cells) was verified. IMDM was supplemented with 10% BIT serum replacement as the basal culture medium for hematopoietic stem cells (HSC), and three growth factors necessary for maintaining stem cell proliferation were added (10ng / mL SCF, 100ng / mL TPO, 100ng / mL FILT3). After 3 days of this basal culture, the 10% THY supplement was added to the basal culture medium and the three cytokines to treat human umbilical cord blood-derived CD34+ hematopoietic stem cells as the experimental group and cultured for 14 days. The control group was added with 10% PBS (as control group 1) and 10% FBS (#10091148; Gibco TM ) (as control group 2). The human umbilical cord blood-derived CD34+ hematopoietic stem cells can be obtained by conventional methods in the prior art (for example, see CN114058584A) and enriched using CD34 microbeads magnetic beads (#130-046-702; Miltenyi) according to the reagent instructions. Figure 13 The figure shows microscopic observation of HSC morphology after 14 days of culture with calf thymus supernatant (THY). Cell counts were performed using a CountStar cell counter and analyzed graphically. The results demonstrate that calf thymus supernatant (THY) significantly increased the total number of hematopoietic stem cells expanded in vitro compared to both control groups 1 (PBS) and 2 (FBS).

[0126] Example 14: Effect of Calf Thymus Supernatant (THY) on Hematopoietic Stem Cell Colony Formation Ability

[0127] Currently, in vitro cloning assays for hematopoietic stem cells and early progenitor cells primarily utilize CFU colony formation assays, the gold standard for in vitro testing of hematopoietic stem cell function. By simulating the in vivo hematopoietic environment and process, the proliferation and differentiation of hematopoietic stem cells can be observed in vitro, thereby determining their ability to generate hematopoietic progenitor cells and differentiate into multiple hematopoietic cell lineages, indirectly reflecting the presence of hematopoietic stem cells.

[0128] In this example, the THY extract obtained in Example 1 was used as an additive component of the hematopoietic stem cell (HSC) culture medium to obtain HSCs under three culture conditions: control group 1, basal medium StemSpan TM SFEM (#09600; Stemcell Technologies) + 10% PBS; control group 2, basal medium StemSpan TMSFEM (#09600; Stemcell Technologies) + 10% FBS; and experimental group, StemSpan TM SFEM + 10% THY. HSC cells obtained under three culture conditions were subjected to CFU colony formation assay to compare their stem cell stemness. The CFU assay used methylcellulose semi-solid medium MethoCult TM (#H4330; Stemcell Technologies) is used as the differentiation medium culture condition. Hematopoietic stem cells cultured under the two different conditions will proliferate and differentiate during the culture period, generating mature blood cell colonies. The function and quality of hematopoietic stem cells are identified and analyzed based on the number and type of colonies. CFU colonies are generally divided into: erythroid colony-forming units (CFU-E), erythroid colony-forming units (BFU-E), granulocyte colony-forming units (CFU-G), macrophage colony-forming units (CFU-M), granulocyte / macrophage forming units (CFU-GM), and mixed cell line colony-forming units (CFU-GEMM).

[0129] Figure 14 The results showed that HSCs cultured with calf thymus supernatant (THY) generated greater numbers of CFU-G, BFU-E, and CFU-GEMM in CFU formation experiments compared to control group 1 (HSCs cultured with PBS) and control group 2 (HSCs cultured with FBS). These results suggest that calf thymus supernatant (THY) expands a greater number of hematopoietic stem cells with differentiation potential.

[0130] Example 15 Effect of Calf Thymus Supernatant THY on T Cell Expansion

[0131] Furthermore, in order to verify the excellent effect of the thymus supernatant THY obtained in the present invention, the thymus supernatant THY of the present invention was compared with thymopentin, thymosin α, and thymosin β4 of thymus extract in the prior art to compare their effects on T cell proliferation.

[0132] The control group "PBS" culture medium composition was ImmunoCult-XF medium (#10981; STEMCELL Technology), 10 ng / mL IL7 / IL15, and 10% PBS buffer by volume. Dynabeads were added to this culture medium every three days. TMHuman T-Activator CD3 / CD28 magnetic beads (#11132D; Thermo Fisher) were used as T cell activators. The culture medium composition of experimental group 1 "THY" was based on the control group culture medium, with PBS replaced and 2 mg / mL of the above-mentioned newborn calf thymus supernatant THY added. The culture medium of experimental group 2 "TP5" was based on the control group culture medium, with PBS replaced and 1 mg / mL TP5 (thymopentin; #T2598; TargetMol) added. The culture medium of experimental group 3 "Tα1" was based on the control group culture medium, with PBS replaced and 0.1 mg / mL Tα1, also known as thymosin α1 (Thymosin α1; #TP1029; TargetMol) added. The culture medium of experimental group 4 "Tβ4" was based on the control group culture medium, with PBS replaced and 0.1 mg / mL Tβ4 (Thymosin β4; #TP2319; TargetMol) added. The working concentrations of thymopentin, thymosin α, and thymosin β4 were determined based on the reagent company's instructions and the working concentrations determined in published literature by users of related products. Peripheral blood-derived T cells were cultured in the five culture media described above for 14 days. The total cell count was recorded using a Countstar cell analyzer, and the cell expansion fold was analyzed and plotted. ** indicates a p-value less than 0.01.

[0133] like Figure 15 As shown, it can be seen that Tα1 does not affect the in vitro expansion of T cells, but TP5 and Tβ4 have an inhibitory effect on T cell expansion. THY in the present invention has a stronger expansion effect on T cells than thymopentin, thymosin alpha-sin and thymosin β4 in the prior art.

[0134] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for culturing or expanding immune cells or progenitor cells thereof in vitro, comprising adding an extracellular extract of thymus tissue to a cell culture medium; The thymus tissue is obtained from a newborn mammal; The newborn mammal is a cow; The preparation method of the thymus tissue extracellular extract comprises: adding a buffer to the thymus tissue to dissolve the thymus tissue extracellular substances in the buffer, and collecting the supernatant by centrifugation; The centrifugation includes first low-temperature low-speed centrifugation and then low-temperature high-speed centrifugation; The immune cells are T cells; The immune cell progenitor cells are hematopoietic stem cells; The low-temperature low-speed centrifugation is 600-4000 rpm, 2-10°C, and centrifugation for 5-15 minutes; The low-temperature high-speed centrifugation is 6000-12000 rpm, 2-10° C., and centrifugation for 20-40 minutes.

2. The method according to claim 1, wherein: When culturing or expanding T cells in vitro, the cell culture medium further comprises IL7 and IL15; The concentrations of IL7 and IL15 are 5-15 ng / ml respectively; The basal medium of the cell culture medium is ImmunoCult-XF medium.

3. The method according to claim 1, wherein: When culturing or expanding hematopoietic stem cells in vitro, the cell culture medium further comprises 5-15 ng / ml SCF, 80-120 ng / ml TPO, and 80-120 ng / ml FILT3; The basal culture medium of the cell culture medium is IMDM culture medium.

4. The method according to claim 1, wherein: The low-temperature low-speed centrifugation is 800-3000 rpm, 2-8°C, and centrifugation for 5-15 minutes; The low-temperature high-speed centrifugation is performed at 6000-12000 rpm, 2-8° C., and for 20-40 minutes.

5. A cell culture medium comprising an extracellular extract of thymus tissue; The thymus tissue is obtained from a newborn mammal; The newborn mammal is a cow; The preparation method of the thymus tissue extracellular extract comprises: adding a buffer to the thymus tissue to dissolve the thymus tissue extracellular substances in the buffer, and collecting the supernatant by centrifugation; The centrifugation includes first low-temperature low-speed centrifugation and then low-temperature high-speed centrifugation; in the preparation method of the thymus tissue extracellular extract, the low-temperature low-speed centrifugation is 600-4000 rpm, 2-10°C, and centrifugation for 5-15 minutes; The low-temperature high-speed centrifugation is 6000-12000 rpm, 2-10° C., and centrifugation for 20-40 minutes.

6. The cell culture medium according to claim 5, wherein: In the method for preparing the extracellular extract of thymus tissue, the low-temperature low-speed centrifugation is 800-3000 rpm, 2-8°C, and centrifugation for 5-15 minutes; The low-temperature high-speed centrifugation is performed at 6000-12000 rpm, 2-8°C, and for 20-40 minutes.

7. The cell culture medium according to claim 5, wherein: The content of the thymus tissue extracellular extract is 5%-20% by volume.

8. The cell culture medium according to claim 7, wherein: The cell culture medium further comprises IL7 and IL15.

9. The cell culture medium according to claim 8, wherein: The concentrations of IL7 and IL15 were 5-15 ng / ml, respectively.

10. The cell culture medium according to claim 9, wherein: The concentrations of IL7 and IL15 were 8-12 ng / ml, respectively.

11. The cell culture medium according to claim 8, wherein: The basal culture medium of the cell culture medium is 1640 culture medium, 1640+β-ME culture medium, αMEM culture medium, LonzaX-vivo15 culture medium or ImmunoCult-XF culture medium.

12. The cell culture medium according to claim 11, wherein: The basal culture medium is ImmunoCult-XF medium.

13. The cell culture medium according to claim 7, wherein: The cell culture medium further comprises 5-15 ng / ml SCF, 80-120 ng / ml TPO, and 80-120 ng / ml FILT3.

14. The cell culture medium according to claim 13, wherein: The basal culture medium of the cell culture medium is IMDM culture medium.

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