In vitro induction composition and culture method of umbilical cord blood-derived gamma delta T cells

Abstract

The present application belongs to the field of cell biology, and particularly relates to an in vitro induction composition of umbilical cord blood derived gamma delta T cells and an in vitro induction culture method. The present application is directed to inducing differentiation of CD34+ hematopoietic stem cells to form gamma delta T cells, obtaining a large amount of hematopoietic stem cells by in vitro sorting expansion of CD34+, and then inducing differentiation to form gamma delta T cells by using a suitable induction culture medium, and proliferating and culturing by using a suitable in vitro proliferation culture medium. The method can realize efficient expansion of umbilical cord blood derived gamma delta T cells, significantly improve the expansion capacity of gamma delta T cells, effectively solve the problem of insufficient expansion of umbilical cord blood gamma delta T cells, and can meet the clinical use requirements.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, specifically relating to an in vitro induction composition for umbilical cord blood-derived γδT cells and an in vitro induction culture method. Background Technology

[0002] Umbilical cord blood, as a biological resource rich in hematopoietic stem cells and immune progenitor cells, contains γδT cells that, due to their low immunogenicity and strong tumor-invasive ability, have become an important research subject in the field of tumor immunotherapy. γδT cells are a unique subset of T cells whose TCRs are composed of γ and δ chains, enabling them to recognize tumor-associated antigens in a non-MHC-restricted manner, exhibiting characteristics of both innate and adaptive immunity. Studies have shown that, compared to γδT cells derived from peripheral blood, umbilical cord blood-derived γδT cells are not only younger but also more functionally primitive.

[0003] However, the proportion of γδT cells in cord blood is extremely low, usually less than 1% of T cells, far lower than the 1%-5% γδT cell content in peripheral blood. Furthermore, cord blood γδT cells are predominantly Vδ1 subsets, while peripheral blood γδ2 subsets (Vγ9Vδ2) are dominant. The Vδ2 subset can directly activate the TCR signaling pathway through phosphorylated antigens (such as IPP or zoledronic acid), while the Vδ1 subset has a weaker response to conventional stimuli and requires activation and expansion by tumor-associated lipid antigens. Its efficiency is limited by donor variability, subset heterogeneity, and limitations in in vitro culture technology, severely restricting its clinical application.

[0004] Currently, methods for expanding peripheral blood γδT cells are relatively mature. These methods involve collecting peripheral blood from adults, isolating mononuclear cells, activating them with phosphoantigens (such as HMBPP and IPP) or bisphosphonates (such as Zoledronic acid), and then expanding them using cytokines such as IL-2 and IL-15. This approach can obtain a large number of γδT cells within 10-14 days and has been used in several early clinical studies for cell therapy. However, the content of γδT cells in umbilical cord blood is much lower than in peripheral blood, and it is mainly composed of the Vδ1 subset, which has a weak response to phosphoantigen stimulation. Expanding these cells using zoledronic acid and cytokines results in a small number of cells with low purity. Additionally, researchers have attempted to combine magnetic bead sorting with artificial antigen-presenting cells (aAPCs) for expansion. This involves isolating γδT cells from umbilical cord blood mononuclear cells using magnetic beads, and then stimulating them with artificial antigen-presenting cells (such as K562 cells) and anti-CD3 antibodies to promote the expansion of Vδ1+ T cells. However, the mechanism of action of Vδ1+ T cells is not yet fully understood. In addition, EBV-LCL has been used as feeder cells, specifically by expanding different subtypes of γδ T cells in vitro using feeder cells composed of irradiated PB mononuclear cells (PBMCs) and Epstein-Barr virus-lymphoblastic cell lines (EBV-LCLs). However, using artificial antigen-presenting cells or EBV-LCLs as feeder cells poses certain safety risks and may introduce potential uncontrollable hazards.

[0005] In summary, current techniques for expanding umbilical cord blood γδT cells primarily rely on magnetic bead sorting and trophoblast cell co-culture. However, the extremely low concentration of γδT cells in umbilical cord blood results in a low initial cell quantity for expansion, insufficient to meet the needs of clinical treatment. Furthermore, while trophoblast cell co-culture can increase the γδT cell expansion rate to some extent, the introduced trophoblast cells may affect the safety and immunoreactivity after cell reinfusion. Therefore, the field anticipates developing a safe and efficient in vitro induction and culture method for umbilical cord blood-derived γδT cells, which would be of significant importance for the application of umbilical cord blood-derived γδT cells. Summary of the Invention

[0006] Therefore, the first objective of this invention is to provide an in vitro induction composition for umbilical cord blood-derived γδT cells, wherein the in vitro induction composition amplifies γδT cell pure factors, thereby achieving in vitro induction of umbilical cord blood-derived γδT cells, and has the advantages of safety and high efficiency.

[0007] The second objective of this invention is to provide an in vitro induction and culture method for umbilical cord blood-derived γδT cells. This method can achieve efficient expansion of umbilical cord blood-derived γδT cells at relatively low concentrations, effectively solving the problem of insufficient expansion of umbilical cord blood γδT cells and meeting clinical needs.

[0008] To address the aforementioned technical problems, this invention provides an in vitro induction composition for umbilical cord blood-derived γδT cells, comprising the following components:

[0009] IL-7 1-20 parts by weight;

[0010] Differentiation inducer 0.5-2 parts by weight;

[0011] Notch signaling agonist 1000-3000 parts by weight;

[0012] 0.05-0.2 parts by volume of plasma;

[0013] The units for the weight parts and volume parts are ng and mL, respectively.

[0014] Specifically, in the in vitro induction composition for umbilical cord blood-derived γδT cells:

[0015] The differentiation inducer includes transforming growth factor and / or RORγt agonist; and / or,

[0016] The Notch signaling agonist includes recombinant human DLL4 protein and / or thymic stromal lymphopoietin (TSLP).

[0017] Preferably, the transforming growth factor includes TGF-β1;

[0018] Preferably, the RORγt agonist includes RORγt agonist 2, i.e., RORγt agonist 2, with the molecular formula C2. 30 H 30 F3N3O4S, Product CAS No.: 2663787-92-4.

[0019] The present invention also provides a preferred in vitro induction composition for umbilical cord blood-derived γδT cells, comprising the following components:

[0020] IL-7 1-20 parts by weight;

[0021] TGF-β1 0.5-2 parts by weight;

[0022] 1000-3000 parts by weight of recombinant human DLL4 protein;

[0023] 0.05-0.2 parts by volume of plasma;

[0024] The units for the weight parts and volume parts are ng and mL, respectively.

[0025] This invention also discloses an in vitro induction culture medium for umbilical cord blood-derived γδT cells, comprising a basal culture medium and an in vitro induction composition for the umbilical cord blood-derived γδT cells; wherein,

[0026] The concentration of IL-7 based on the basal culture medium is 1-20 ng / mL, preferably 5-15 ng / mL, more preferably 10 ng / mL; and / or,

[0027] The differentiation inducer is present at a concentration of 0.5-2 ng / mL based on the basal culture medium, preferably 1-1.5 ng / mL, more preferably 1 ng / mL; and / or,

[0028] The Notch signaling agonist is present at a concentration of 1-3 μg / mL, preferably 1.5-2.5 μg / mL, more preferably 2 μg / mL, based on the basal culture medium; and / or,

[0029] The plasma concentration based on the basal culture medium is 5-20% v / v, preferably 8-15% v / v, and more preferably 10% v / v.

[0030] It should be noted that IL is an abbreviation for interleukin, a class of cytokines produced by immune cells (such as lymphocytes and macrophages) and acting on other cells. IL-7 refers to interleukin-7 that binds to the IL-7 receptor. In the following embodiments of this invention, "IL" refers to interleukin, and different numbers only indicate binding to different receptors.

[0031] Furthermore, among the differentiation inducers, TGF-β1, or Transforming Growth Factor-beta 1, is a multifunctional cytokine belonging to the TGF-β superfamily. It is widely expressed in various cell types, including immune cells, epithelial cells, and mesenchymal cells. It plays a crucial role in cell proliferation, differentiation, apoptosis, immune regulation, and tissue repair.

[0032] Among the differentiation inducers, the RORγt agonist (Retinoic Acid Receptor-RelatedOrphan Receptor Gamma t Agonist) is a small molecule agonist that acts on the transcription factor RORγt, specifically activating key signaling pathways regulating T cell fate. RORγt is a splice variant of the nuclear receptor transcription factor RORγ, primarily expressed in immune cells, playing a central regulatory role, especially in the differentiation of γδT cells and Th17 cells. Among the Notch signaling agonists, the recombinant human DLL4 protein is a human Delta-like ligand 4 (DLL4) protein expressed and purified in vitro using genetic engineering techniques. DLL4 is one of the key ligands in the Notch signaling pathway, playing an important role in angiogenesis, cell differentiation, immune regulation, and tumorigenesis.

[0033] In the Notch signaling agonist, thymic stromal lymphopoietin (TSLP) is a cytokine similar to IL-7 that plays a key role in immune regulation, inflammatory responses, and allergic diseases. TSLP is primarily produced by epithelial cells, keratinocytes, and stromal cells, and exerts its effects by binding to the TSLP receptor (TSLPR).

[0034] In some feasible embodiments, the plasma includes autologous inactivated human plasma or type AB human plasma.

[0035] In some feasible embodiments, the basal culture medium includes serum-free γδT cell basal culture medium.

[0036] This invention also discloses an in vitro induction and culture method for umbilical cord blood-derived γδT cells, comprising the following steps:

[0037] (1) CD34+ cells were sorted from umbilical cord blood and seeded cells were cultured to obtain CD34+ hematopoietic stem cells;

[0038] (2) The CD34+ hematopoietic stem cells were inoculated into the in vitro induction culture medium of the umbilical cord blood-derived γδT cells for induction culture;

[0039] (3) Continue to add in vitro expansion culture medium for expansion culture to obtain the required γδT cells.

[0040] In some feasible embodiments, step (1), the step of sorting CD34+ cells based on umbilical cord blood, includes:

[0041] S1, extract umbilical cord blood mononuclear cells, aspirate the white membrane layer, and resuspend in buffer solution to obtain cell suspension;

[0042] S2, dilute the cell suspension to a cell concentration of 1×10⁻⁶. 8 -5×10 8 cells / mL;

[0043] S3, the diluted cell suspension antibody mixture was incubated with reagents to obtain the sorting system;

[0044] S4, using flow cytometry to perform repeated magnetic bead sorting on the sorting system, i.e., sorted CD34+ hematopoietic progenitor cells.

[0045] In some feasible embodiments, in step (1), the seed cell culture medium for the seed cell culture step includes serum-free stem cell culture medium and hematopoietic stem cell supplement; wherein,

[0046] The serum-free culture medium for stem cells accounts for 80-90 wt% of the total volume of the seed cell culture medium.

[0047] The hematopoietic stem cell supplement accounts for 10-20 wt% of the total amount of the seed cell culture medium.

[0048] Preferably, the suspension density of the CD34+ hematopoietic progenitor cells based on the seed cell culture medium is <5×10⁻⁶. 4 cells / mL;

[0049] Preferably, the temperature for the seed cell culture step is 35-38℃, and the culture period is 5-8 days;

[0050] Preferably, the seed cell culture step further includes the step of replenishing the seed cell culture medium during the culture process.

[0051] In some feasible embodiments, the conditions for the induction culture step in step (2) include:

[0052] Incubation temperature 35-38°C; and / or,

[0053] The culture conditions were as follows: cultured at 5% v / v CO2; and / or,

[0054] The incubation period is 2-3 days;

[0055] Preferably, the seeding density of the CD34⁺ hematopoietic stem cells based on the in vitro induction culture medium is 0.8 × 10⁻⁶. 6 -1.2×10 6 Cells / mL.

[0056] In some feasible embodiments, in step (3), the in vitro amplification culture medium includes the following components at the following concentrations: IL-2 150-250U / mL, IL-15 20-30ng / mL, active drug 3-8μM / mL, and final plasma concentration 3-8% v / v;

[0057] Preferably, the in vitro amplification culture medium comprises the following components at the following concentrations: IL-2 200 U / mL, IL-15 25 ng / mL, active drug 5 μM / mL, and final plasma concentration of 5% v / v.

[0058] In some feasible embodiments, the active pharmaceutical ingredient includes zoledronic acid or bisphosphonates;

[0059] Preferably, the conditions for the amplification culture step include:

[0060] Incubation temperature 35-38°C; and / or,

[0061] The culture conditions were as follows: cultured at 5% v / v CO2; and / or,

[0062] The cultivation time is 10-15 days;

[0063] Preferably, the amplification culture step further includes the step of continuing to add the in vitro amplification culture medium during the amplification culture process.

[0064] The present invention also discloses umbilical cord blood-derived γδT cells prepared according to the method.

[0065] The present invention also discloses the use of the umbilical cord blood-derived γδT cells in the preparation of drugs for the prevention or treatment of diseases;

[0066] The drug in question is an adjunct therapy for cancer.

[0067] Preferably, the cancer is a solid tumor.

[0068] The in vitro induction composition and culture medium for umbilical cord blood-derived γδT cells described in this invention are designed to induce CD34+ hematopoietic stem cells to differentiate into γδT cells. By adding recombinant human DLL4 protein as a Notch signaling agonist and TGF-β1 factor to the induction culture medium, CD34+ cells can be effectively induced to differentiate into T cell lineages, resulting in a large number of γδT cells. This effectively overcomes the problem of insufficient expansion due to the low content of umbilical cord blood γδT cells.

[0069] The in vitro induction and culture method for umbilical cord blood-derived γδT cells described in this invention involves obtaining a large number of hematopoietic stem cells through in vitro sorting and expansion of CD34+ cells, followed by induction differentiation into γδT cells using a suitable induction culture medium, and then proliferation culture using a suitable in vitro proliferation culture medium. This method not only achieves efficient expansion of umbilical cord blood-derived γδT cells at relatively low concentrations, significantly improving the expansion capacity of γδT cells and effectively solving the problem of insufficient expansion of umbilical cord blood γδT cells, but also, by using pure factors for expansion, effectively avoids interference from trophoblast cells, ensuring the safety of cell reinfusion and providing a more reliable solution for clinical applications, thus meeting clinical needs. Attached Figure Description

[0070] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0071] Figure 1 This is a flowchart illustrating the in vitro expansion process of umbilical cord blood γδT cells according to the present invention.

[0072] Figure 2 This is a morphological image of the umbilical cord blood γδT cells amplified in Example 3 of the present invention;

[0073] Figure 3 The results are based on the counting of 100 μL of cell suspension taken at each time point under the schemes of Examples 3-9 and Comparative Examples 1-3. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0075] In the following embodiments of the present invention, the units for all added components conform to conventional definitions in the art. For example, mass ratio, volume ratio, etc., are all unit definitions conventionally recognized in the art. Furthermore, U stands for "Unit," a standardized measurement method for bioactive substances, indicating a defined number of units of a component per milliliter of solution. For example, a concentration of 150-250 U / mL of IL-2 in the in vitro amplification culture medium means that each milliliter of solution contains 150-250 units of IL-2 activity.

[0076] In the following embodiments of the present invention, in order to achieve in vitro induction culture of umbilical cord blood-derived γδT cells, an in vitro induction composition for umbilical cord blood-derived γδT cells is provided, comprising the following components:

[0077] IL-7 1-20 parts by weight;

[0078] TGF-β1 0.5-2 parts by weight;

[0079] 1000-3000 parts by weight of recombinant human DLL4 protein;

[0080] 0.05-0.2 parts by volume of plasma;

[0081] The units for the weight parts and volume parts are ng and mL, respectively.

[0082] As a feasible implementation, the plasma includes autologous inactivated human plasma or AB type human plasma.

[0083] In the in vitro induction composition for umbilical cord blood-derived γδT cells described in this invention, DLL4 protein is selected as a Notch signaling agonist, which can effectively induce CD34+ to differentiate into T cell lineages, providing a basic guarantee for the efficient expansion of CD34+ cells; the added TGF-β1 ensures the growth-promoting state during the induction process, enabling efficient and safe in vitro induction of γδT cells even when the amount of umbilical cord blood-derived γδT cells is low.

[0084] In the following embodiments of the present invention, to adapt to the in vitro induction culture protocol of umbilical cord blood-derived γδT cells, an in vitro induction culture medium for umbilical cord blood-derived γδT cells is also provided, comprising a basal culture medium and the in vitro induction composition for umbilical cord blood-derived γδT cells; wherein,

[0085] The concentration of IL-7 based on the basal culture medium is 1-20 ng / mL, preferably 5-15 ng / mL, more preferably 10 ng / mL; and / or,

[0086] The TGF-β1 concentration based on the basal culture medium is 0.5-2 ng / mL, preferably 1-1.5 ng / mL, more preferably 1 ng / mL; and / or,

[0087] The recombinant human DLL4 protein concentration based on the basal culture medium is 1-3 μg / mL, preferably 1.5-2.5 μg / mL, more preferably 2 μg / mL; and / or,

[0088] The plasma concentration based on the basal culture medium is 5-20% v / v, preferably 8-15% v / v, and more preferably 10% v / v.

[0089] As a feasible implementation, the basal culture medium, including serum-free γδT cell basal culture medium, can be a commercially available culture medium. As an exemplary implementation, in the following embodiments of the present invention, γδT cell basal culture medium (γδT cells Basal Medium, catalog number IMC-015-BM), HiXpan™ γδT cell serum-free culture medium (catalog number GD990-M), human γ / δT cell (Clin-SFM®) culture medium (catalog number 1005), or other conventional and universal γδT cell serum-free basal culture media in the art can be selected.

[0090] In the following embodiments of the present invention, in order to realize the in vitro induction culture protocol for umbilical cord blood-derived γδT cells, a method for in vitro induction culture of umbilical cord blood-derived γδT cells is also provided, comprising the following steps:

[0091] (1) CD34+ cells were sorted from umbilical cord blood and seeded cells were cultured to obtain CD34+ hematopoietic stem cells;

[0092] (2) The CD34+ hematopoietic stem cells were inoculated into the in vitro induction culture medium of the umbilical cord blood-derived γδT cells for induction culture;

[0093] (3) Continue to add in vitro expansion culture medium for expansion culture to obtain the required γδT cells.

[0094] As a feasible implementation scheme, step (1), the step of sorting CD34+ cells based on umbilical cord blood includes:

[0095] S1, extract umbilical cord blood mononuclear cells, aspirate the white membrane layer, and resuspend in buffer solution to obtain cell suspension;

[0096] S2, dilute the cell suspension to a cell concentration of 1×10⁻⁶. 8 -5×10 8 cells / mL;

[0097] S3, the diluted cell suspension antibody mixture was incubated with reagents to obtain the sorting system;

[0098] S4, using flow cytometry to perform repeated magnetic bead sorting on the sorting system, i.e., sorted CD34+ hematopoietic progenitor cells.

[0099] As a feasible implementation, the buffer solution can be selected from buffer solutions commonly used in the field for cell separation.

[0100] As an exemplary embodiment, the step of sorting CD34+ cells based on umbilical cord blood according to the present invention may include the following operational steps:

[0101] S1. Mononuclear cells were extracted from umbilical cord blood. The cells were washed and centrifuged using standard methods, and the white membrane layer was aspirated. After routine laboratory washing, the white membrane layer was resuspended in approximately 1 mL of buffer and thoroughly mixed (avoiding excessive foaming).

[0102] S2, take a new EP tube, add 990 μL of buffer, and add 10 μL of cell suspension to the buffer (100-fold dilution); take another new EP tube, add 10 μL of nucleated cell counting solution, then add 10 μL of 100X (100-fold) diluted cell suspension, mix thoroughly, and count using a Countess counter (remove trypan blue dilution factor); calculate the total cell count based on the count results; if the total cell count is below 1 × 10⁻⁶... 7 -10×10 7 The cells need to be resuspended at 1×10⁻⁶. 8 / mL concentration, such as cells at 1×10 8 -5×10 8 If the cells are between 5 × 10⁶, resuspend in 1 mL buffer without adjustment. 8 Therefore, sorting needs to be done in multiple stages;

[0103] S3. Transfer the cell suspension into a sterile capped polystyrene flow cytometer and add 100 μL / mL of antibody mixing reagent to the cell suspension. Do not shake or pipette vigorously, as it does not need to be mixed on its own. After mixing, incubate at room temperature (15℃-25℃) for 8-12 minutes. After the procedure, thoroughly mix the RapidSpheres™ magnetic beads until there is no obvious retention or precipitation on the bottom and sidewalls when inverted and shaken, and the liquid is a smooth, uniform brownish-brown liquid. Add 75 μL / mL to the cell suspension, mix well, and incubate at room temperature (15℃-25℃) for 3-8 minutes.

[0104] S4. Bring the sorting system to a fixed volume and insert it into the purple magnetic pole (ensure the magnetic pole is upright, aligning with the text on the magnet). Insert it all the way in and incubate at room temperature (15℃-25℃) for 2-4 minutes. Lift the magnet and pour out the supernatant at a suitable angle, holding this angle for 1-2 seconds. Do not bump or shake. Then remove the flow cytometry tube, add buffer to a fixed volume, and mix 2-3 times (avoid excessive foaming). Insert the magnet again using the same method and incubate at room temperature (15℃-25℃) for 2-4 minutes. Repeat the above steps. After removing the flow cytometry tube for the last time, you can proceed to the subsequent CD34+ amplification culture stage.

[0105] In some feasible embodiments, in step (1), the seed cell culture medium for the seed cell culture step includes serum-free stem cell culture medium and hematopoietic stem cell supplement; wherein,

[0106] The serum-free culture medium for stem cells accounts for 80-90 wt% of the total volume of the seed cell culture medium.

[0107] The hematopoietic stem cell supplement accounts for 10-20 wt% of the total amount of the seed cell culture medium.

[0108] Preferably, the mass ratio of the serum-free stem cell culture medium to the hematopoietic stem cell supplement is 9:1, that is, the serum-free stem cell culture medium accounts for 90 wt% of the total seed cell culture medium, and the hematopoietic stem cell supplement accounts for 10 wt% of the total seed cell culture medium.

[0109] As an exemplary embodiment, the hematopoietic stem cell supplement is selected from CD34+ amplification supplements commonly used in the art.

[0110] As an exemplary embodiment, the seed cell culture medium is prepared by mixing StemSpan™ SFEM II medium and StemSpan™ CD34+ amplification supplement (10-fold dilution). Other culture media and supplements that are foreseeable in the art may also be used for cell culture.

[0111] As an exemplary implementation, in the following embodiment of the present invention, the CD34+ amplification supplement is Stemcell brand StemSpan™ CD34+ inducing amplification factor, product code 02691.

[0112] As an exemplary embodiment, the CD34+ hematopoietic progenitor cells have a suspension density of <5×10⁻⁶ in the seed cell culture medium. 4 cells / mL;

[0113] Preferably, the temperature for the seed cell culture step is 35-38℃, and the culture period is 5-8 days;

[0114] Preferably, the seed cell culture step further includes the step of replenishing the seed cell culture medium during the culture process.

[0115] As an exemplary embodiment, the seed cell proliferation culture step of the present invention may include the following operational steps:

[0116] Day 0: Prepare CD34+ seed cell culture medium. Mix pre-thawed StemSpan™ SFEM II medium with StemSpan™ CD34+ amplification supplement (10-fold dilution). The prepared complete medium can be stored at 2-8℃ for one week. Calculate the total number of cells obtained from the first part of the counting and sorting. Use the prepared medium at a ratio of <5×10⁻⁶ cells / cells. 4CD34+ hematopoietic progenitor cells were resuspended at a density of 1 cell / mL; then, after gently pipetting to mix, 5 mL was seeded into a T25 culture flask, labeled, and incubated at 37°C for 48 h.

[0117] Day 2: Change the culture medium as follows: Transfer all the culture medium and culture in the T75 culture flask to a centrifuge tube and centrifuge to remove the supernatant; then gently mix with fresh culture medium by pipetting, re-spread it onto the T25 culture flask, label it, and incubate it in a 37℃ incubator for 48 hours.

[0118] Day 4: Count the number of cells using nucleated cell counting solution (specific method: take a small amount of cells, dilute with nucleated cell counting solution 1:1, and estimate the final concentration to be around 10). 6 (At the / mL level, avoid exceeding the range of the counters) Adjust cell density to <5×10⁹ / mL. 4 After achieving a cell / mL ratio, re-plate the culture into flasks. Transfer all the culture medium and culture from T25 to centrifuge tubes, centrifuge, and remove the supernatant; gently mix with an appropriate amount of fresh complete culture medium by pipetting, then plate the culture onto T25 flasks, label them, and incubate at 37°C for 2 days.

[0119] Day 6: Harvest CD34+ cells. If a larger volume of γδT cells is needed, continue culturing to obtain more seed cells.

[0120] In some feasible embodiments, the conditions for the induction culture step in step (2) include:

[0121] Incubation temperature 35-38℃; and / or,

[0122] The culture conditions were as follows: cultured at 5% v / v CO2; and / or,

[0123] The incubation period is 2-3 days;

[0124] Preferably, the seeding density of the CD34⁺ hematopoietic stem cells based on the in vitro induction culture medium is 0.8-1.2 × 10⁻⁶. 6 Cells / mL.

[0125] In some feasible embodiments, in step (3), the in vitro amplification culture medium includes the following components at the following concentrations: IL-2 150-250U / mL, IL-15 20-30ng / mL, active drug 3-8μM / mL, and final plasma concentration 3-8% v / v;

[0126] Preferably, the in vitro amplification culture medium comprises the following components at the following concentrations: IL-2 200 U / mL, IL-15 25 ng / mL, active drug 5 μM / mL, and final plasma concentration of 5% v / v.

[0127] In some feasible embodiments, the active pharmaceutical ingredient includes zoledronic acid (ZOL).

[0128] Preferably, the conditions for the amplification culture step include:

[0129] Incubation temperature 35-38°C; and / or,

[0130] The culture conditions were as follows: cultured at 5% v / v CO2; and / or,

[0131] The cultivation time is 10-15 days;

[0132] Preferably, the amplification culture step further includes the step of continuing to add the in vitro amplification culture medium during the amplification culture process.

[0133] As an exemplary embodiment, the in vitro induction culture and in vitro amplification culture steps of the present invention may include:

[0134] Day 0: Prepare the in vitro induction culture medium described above, resuspend CD34⁺ hematopoietic stem cells, and adjust their seeding density to 1 × 10⁻⁶. 6 Cells / mL, the cells were seeded into T75 cell culture flasks and cultured at 37°C and 5% v / v CO2;

[0135] Day 3: Add 20-40 mL of the amplification medium described above, and continue culturing after replenishment;

[0136] Day 5: Add 80-120 mL of the amplification medium described above, transfer all cells and culture medium to a T175 culture flask, and continue culturing;

[0137] Day 7: Add 280-320 mL of amplification culture medium and transfer the cell system to an air-permeable cell culture bag;

[0138] Day 10: Add another 530-580 mL of amplification medium and continue culturing;

[0139] Day 12: Add 900-1100 mL of amplification medium (without plasma) and continue culturing;

[0140] Day 14: Collect suspended cells from the culture medium and perform quality tests, such as flow cytometry, sterility testing, mycoplasma testing, and endotoxin testing.

[0141] In the following embodiments of the present invention, the above-described in vitro induction culture method is used to efficiently expand umbilical cord blood-derived γδT cells, thereby realizing the utilization of γδT cells.

[0142] Example 1

[0143] As attached Figure 1 The process shown in this embodiment involves sorting CD34+ cells based on umbilical cord blood.

[0144] In this embodiment, the buffer used for cell sorting is STEMCELL's EasySep™ brand, product code #20144.

[0145] In this embodiment, the nucleated cell counting solution used for cell counting is selected from STEMCELL Technologies, product number #07060.

[0146] Remove the selection buffer from the refrigerator and allow it to return to room temperature (15℃-25℃).

[0147] Following standard procedures, mononuclear cells were extracted from umbilical cord blood, washed routinely, and then subjected to density gradient centrifugation to aspirate the white membrane layer.

[0148] After routine laboratory washing, the obtained white film layer was resuspended in 1 mL of the aforementioned room temperature buffer and thoroughly mixed (avoiding excessive foaming).

[0149] Take a new EP tube, add 990uL of buffer, and add 10uL of the aforementioned cell suspension to the buffer (100-fold dilution).

[0150] Take another new EP tube, add 10 uL of nucleated cell counting solution, then add 10 uL of 100-fold diluted cell suspension, mix thoroughly, and count using a Countess counter (remove trypan blue dilution factor).

[0151] Calculate the total cell count based on the counting results. If the total cell count is 1 × 10⁻⁶... 7 -10×10 7 The cells need to be resuspended at 1×10⁻⁶. 8 / mL concentration, such as cells at 1×10 8 -5×10 8 If the cells are between 5 × 10⁶, resuspend in 1 mL buffer without adjustment. 8 Therefore, sorting needs to be done in stages.

[0152] Transfer the cell suspension into a 5 mL sterile capped polystyrene flow cytometer (75 mm × 12 mm), and add 100 μL / mL Selection Cocktail (Selection cocktail is an antibody mixing reagent; do not shake or blow vigorously, and it does not need to be mixed on its own). Mix well and incubate at room temperature (15℃-25℃) for 10 minutes.

[0153] Mix RapidSpheres™ magnetic beads thoroughly until there is no obvious retention or precipitation on the bottom and sidewalls when inverted and shaken, and the liquid is a smooth, uniform brownish-brown liquid. Add 75 uL / mL to the cell suspension, mix well, and incubate at room temperature (15℃-25℃) for 5 minutes.

[0154] Bring the sorting system to a final volume of 2.5 mL. Do not cap the container. Insert the container into the purple magnet (make sure the magnet is upright, aligning with the text on the magnet) and insert it all the way in.

[0155] Incubate at room temperature (15℃-25℃) for 3 minutes. Then, lift the magnet and pour out the supernatant at the following angle, holding it at this angle for 1-2 seconds. Do not bump or shake.

[0156] Remove the flow cytometer tube, add 2.5 mL of buffer, mix 2-3 times (avoid excessive foaming), insert the magnet again using the same method, and incubate at room temperature (15℃-25℃) for 3 minutes.

[0157] Repeat the sorting steps above four times, for a total of 5 x 3 minutes of sorting. After the final removal of the flow cytometry tube, the CD34+ amplification culture stage can begin.

[0158] Example 2

[0159] As attached Figure 1 As shown in the process, this embodiment is based on the cell expansion of the umbilical cord blood CD34+ cells sorted in the aforementioned embodiment 1.

[0160] Day 0 of culture: Prepare the complete culture medium SFEM II+CD34+ supplement required for CD34+ culture. This is achieved by mixing 9 mL of pre-thawed StemSpan™ SFEM II medium and 1 mL of StemSpan™ CD34+ Expansion Supplement (10X) in a 15 mL centrifuge tube at a 9:1 mass ratio. Estimate the amount needed for subsequent use according to the ratio. The prepared complete culture medium can be stored at 2-8℃ for one week.

[0161] Calculate the total number of cells obtained from the first part of counting and sorting, and use the prepared CD34+ amplification complete medium at a ratio of <5×10⁻⁶. 4CD34+ hematopoietic progenitor cells were resuspended at a density of 1 cell / mL. After gently mixing by pipetting, 5 mL was seeded into a T25 culture flask, labeled, and incubated at 37°C for 48 h.

[0162] Day 2 of culture: Change the culture medium as follows: Transfer all the culture medium and culture from the T75 culture flask to a 15mL centrifuge tube, centrifuge at 300g for 5min, and remove the supernatant. Then gently mix with 5mL of fresh CD34+ complete culture medium by pipetting, re-plate onto the T25 culture flask, label it, and incubate at 37℃ for 48h.

[0163] Day 4 of culture: Count the number of cells using nucleated cell counting solution (specific method: aspirate a small amount of cells, dilute with nucleated cell counting solution 1:1, and estimate the final concentration to be around 10). 6 (At the / mL level, avoid exceeding the range of the counters) Adjust cell density to <5×10⁹ / mL. 4 After achieving a cell / mL ratio, re-column the culture in the T25 culture vessel. Transfer all the culture medium and culture from the T25 vessel to 15mL centrifuge tubes, centrifuge at 300g for 5min, and remove the supernatant. Gently mix with an appropriate amount of fresh complete culture medium by pipetting, then plate 5mL of the mixture onto T25 vessels, label them, and incubate at 37℃ for 2 days.

[0164] Day 6 of culture: Harvest CD34+ cells. If a larger volume of γδT cells is needed, culture can continue to obtain more seed cells.

[0165] Example 3

[0166] As attached Figure 1 As shown in the process, this embodiment is based on the seed cells expanded in the aforementioned embodiment 2 for in vitro induction culture and in vitro expansion culture of γδT cells.

[0167] Day 0: Preparation of Induction Medium: Take 20 mL of serum-free basal medium for γδT cells (such as HiXpan™) and add the following components to prepare the induction medium: IL-7: 10 ng / mL, TGF-β1: 1 ng / mL, recombinant human DLL4 protein: 2 μg / mL, and autologous inactivated human plasma: 10% (v / v). Resuspend CD34+ hematopoietic stem cells in the above induction medium and adjust their seeding density to 1 × 10⁻⁶. 6 Cells / mL. Cells were seeded into T75 cell culture flasks and cultured at 37°C and 5% v / v CO2.

[0168] Day 3: Add 30 mL of in vitro amplification medium (5% v / v plasma + 5 μM / mL zoledronic acid (ZOL)). The components of the in vitro amplification medium are as follows: IL-2: 200 U / mL, IL-15: 25 ng / mL. After adding the medium, the total volume of the system is 50 mL. Continue culturing under the aforementioned conditions.

[0169] Day 5: Add another 100 mL of amplification medium (5% v / v plasma), transfer all cells and culture medium to a T175 culture flask, bringing the total culture volume to 150 mL, and continue culturing.

[0170] Day 7: Add another 300 mL of amplification medium, transfer the cell system to an air-permeable cell culture bag, adjust the final plasma concentration to 1% v / v, and the total culture volume to 450 mL.

[0171] Day 10: Continue to add 550 mL of amplification culture medium (1% v / v plasma), bringing the total volume to 1000 mL;

[0172] Day 12: Add another 1000 mL of amplification medium (excluding plasma), for a total culture volume of 2000 mL;

[0173] Day 14: Collect the suspended cells from the culture medium. The morphological image of the umbilical cord blood γδT cells obtained in this embodiment is attached. Figure 2 As shown.

[0174] It can be seen that the expanded γδT cells are round or oval, with an average diameter of 7-10 μm. They easily form cell clusters, and the cell clusters are relatively large, with a diameter of 50-300 μm.

[0175] Example 4

[0176] The in vitro induction and expansion culture steps of γδT cells described in this embodiment are the same as those in Embodiment 3, with the only difference being:

[0177] The induction medium contains different concentrations of the following components: IL-7: 1 ng / mL, TGF-β1: 2 ng / mL, recombinant human DLL4 protein: 1 μg / mL, and autologous inactivated human plasma: 20% v / v.

[0178] The in vitro amplification culture medium contains different concentrations of each component: IL-2: 150 U / mL, IL-15: 30 ng / mL, zoledronic acid (ZOL): 3 μM / mL, and autologous plasma: final concentration 8% v / v.

[0179] Example 5

[0180] The in vitro induction and expansion culture steps of γδT cells described in this embodiment are the same as those in Embodiment 3, with the only difference being:

[0181] The induction medium contained different concentrations of each component: IL-7: 20 ng / mL, TGF-β1: 0.5 ng / mL, recombinant human DLL4 protein: 3 μg / mL, and autologous inactivated human plasma: 5% v / v.

[0182] The in vitro amplification culture medium contains different concentrations of each component: IL-2: 250 U / mL, IL-15: 20 ng / mL, zoledronic acid (ZOL): 8 μM / mL, and autologous plasma: final concentration 3% v / v.

[0183] Example 6

[0184] The in vitro induction and expansion culture steps of γδT cells described in this embodiment are the same as those in Embodiment 3, except that the concentrations of each component in the induction culture medium are different: IL-7: 5 ng / mL, TGF-β1: 1.5 ng / mL, recombinant human DLL4 protein: 1.5 μg / mL, and autologous inactivated human plasma: 15% v / v.

[0185] Example 7

[0186] The in vitro induction and expansion culture steps of γδT cells described in this embodiment are the same as those in Example 3. The only difference is that the concentrations of each component in the induction culture medium are different: IL-7: 15 ng / mL, TGF-β1: 1 ng / mL, recombinant human DLL4 protein: 2.5 μg / mL, and autologous inactivated human plasma: 8% v / v.

[0187] Example 8

[0188] The in vitro induction and expansion culture steps of γδT cells in this embodiment are the same as those in Embodiment 3, except that the recombinant human DLL4 protein in the induction culture medium is replaced with thymic stromal lymphopoietin (TSLP) at a concentration of 15 ng / mL.

[0189] Example 9

[0190] The in vitro induction and expansion culture steps of γδT cells described in this embodiment are the same as those in Embodiment 3, except that TGF-β1 is replaced with RORγt agonist 2 in the induction culture medium at a concentration of 0.5 μM.

[0191] Comparative Example 1

[0192] The in vitro induction and expansion culture steps of the γδT cells described in this comparative example are the same as those in Example 3, except that the induction culture medium does not contain recombinant human DLL4 protein.

[0193] Comparative Example 2

[0194] The in vitro induction and expansion culture steps of the γδT cells described in this comparative example are the same as those in Example 3, except that the induction culture medium does not contain TGF-β1.

[0195] Comparative Example 3

[0196] The in vitro induction and expansion culture steps of γδT cells described in this comparative example are the same as those in Example 3, except that 1 × 10 6 Cells / mL CBMC were seeded into T75 culture flasks.

[0197] Experimental Example

[0198] 1. Flow cytometry detection of γδT cell surface markers

[0199] In this experimental example, the flow cytometry detection method is as follows: First, take 1×10 6 γδT cells were centrifuged at 250g for 5 minutes to remove the supernatant. Then, the cells were washed twice with PBS containing 10% v / v FBS, centrifuged for 5 minutes each time. Next, 2.5 μL of TCRVδ2+ and CD3 antibody was added in the dark, and the cells were incubated on ice for 30 minutes. After incubation, the cells were washed twice again with PBS containing 10% v / v FBS, and centrifuged for 5 minutes each time. The cells were then resuspended in 500 μL of RPMI 1640 complete medium (containing 10% v / v FBS) and filtered through a 70 μm cell strainer to remove aggregated cells. Finally, the cell suspension was analyzed by flow cytometry.

[0200] The flow cytometry results in this experiment are shown in Table 1 below.

[0201] Table 1. Flow cytometry phenotypic results of γδT cells

[0202]

[0203] The results showed that the method of the present invention, by adding recombinant human DLL4 protein as a Notch signaling agonist and adding TGF-β1 factor to the induction culture medium, can effectively induce CD34+ to differentiate into T cell lineages.

[0204] As can be seen from the data results in Examples 3, 8 and Comparative Example 1 above, the addition of recombinant human DLL4 protein or thymic stromal lymphopoietin (TSLP) as Notch signaling agonists in this invention has a better induction effect on CD34+ hematopoietic cells. Compared with the scheme in Comparative Example 1, the induction performance on CD34+ hematopoietic cells is better.

[0205] As can be seen from the data results in Examples 3, 9 and Comparative Example 2 above, the addition of TGF-β1 factor or RORγt agonist in this invention has a better induction effect on CD34+ hematopoietic cells. Compared with the scheme in Comparative Example 2, the induction performance on CD34+ hematopoietic cells is better.

[0206] The data results from Example 3 and Comparative Example 3 show that the purity of CD34+ TCRVδ2+ in Example 3 was significantly higher than that in Comparative Example 3, indicating that direct induction is not conducive to the differentiation of CD34+ into γδT cells.

[0207] 2. Detection of γδT cell killing activity

[0208] The cytotoxic activity of co-cultures of γδT cells collected under the above-mentioned Examples 3-9 and Comparative Examples 1-3 was detected.

[0209] The experiment involved plating target cells (K562) and effector cells (cases 1-3 and control case 1) at effector-to-target ratios of 40:1, 20:1, and 10:1.

[0210] The concentrations of effector cells were 4 × 10⁻⁶. 6 cells / mL, 2×10 6 cells / mL, 1×10 6 Cells / mL, K562 concentration is 1×10 5 100 μL / mL, 3 replicates per group;

[0211] Effector cell spontaneous release wells: 4 × 10 6 cells / mL, 2×10 6 cells / mL, 1×10 6 100 μL / mL, 3 replicates per group;

[0212] Maximum target cell release pore size: 1×10 5 Target cells per mL, 100 μL per well, 3 replicates per group;

[0213] Blank control group: 200 μL of culture medium per well, 3 replicates per group;

[0214] Volume correction control group: 200 uL of culture medium per well, 3 replicates per group. 20 uL of cell lysis buffer was added to each well 45 min before incubation at 37℃ and 5% v / v CO2.

[0215] After plating, centrifuge the culture plate at 250g for 4 min, and incubate it in a 37℃, 5% v / v carbon dioxide incubator for 4 h. 45 min before the end of the incubation, remove the culture plate, centrifuge it at 250g for 4 min, and continue incubation for another 45 min before removing it.

[0216] LDH (lactate dehydrogenase) assay: Centrifuge at 250g for 4 min, aspirate 50 μL of supernatant from each well and transfer to a new 96-well plate. Add 50 μL of substrate solution to each well and incubate at room temperature in the dark for 30 min. Add 50 μL of stop solution to each well, disperse the pigment particles with a shaker, and measure the absorbance at 490 nm.

[0217] In this experimental example, the mean absorbance of the experimental group, the target cell LDH spontaneous release group, and the effector cell group LDH spontaneous release group was subtracted from the mean absorbance of the culture medium blank comparative group to obtain the correction value. The mean absorbance of the target cell maximum LDH release group was subtracted from the mean absorbance of the volume correction group to obtain the correction value.

[0218] In this experimental example, the formula for calculating lethal activity is as follows:

[0219] Activity = (AET) / (T) max -T)×100% (where A: corrected absorbance value of the experimental group, E: corrected absorbance value of the effector cell spontaneous release well, T: corrected absorbance value of the target cell spontaneous release well, T max (Correction value of absorbance for the target cell maximum release group).

[0220] In this embodiment, the detection results are shown in Table 2 below.

[0221] Table 2. Killing activity of γδT cells against K562 cells

[0222]

[0223] The results showed that the addition of recombinant human DLL4 protein as a Notch signaling agonist and the addition of TGF-β1 factor in the induction culture medium of the present invention had a positive correlation between the purity of γδT cells and the killing activity of K562 cells.

[0224] As can be seen from the data results in Examples 3, 8 and Comparative Example 1 above, the addition of recombinant human DLL4 protein or thymic stromal lymphopoietin (TSLP) as Notch signaling agonists in this invention resulted in γδT cells with better purity and killing activity against K562 cells. Compared with the scheme in Comparative Example 1, the purity of γδT cells showed better killing activity against K562 cells.

[0225] As can be seen from the data results in Examples 3, 9 and Comparative Example 2 above, the addition of TGF-β1 factor or RORγt agonist in this invention results in better killing activity of γδT cells against K562 cells. Compared with the scheme in Comparative Example 2, the purity of γδT cells has better killing activity against K562 cells.

[0226] As can be seen from the data results in Example 3 and Comparative Example 3 above, the purity of δT cells under the scheme of Example 3 is positively correlated with the killing activity of K562 cells.

[0227] 3. Amplification factor

[0228] In this experiment, 100 μL of cell suspension was collected at various time points under the protocols of Examples 3-9 and Comparative Examples 1-3 for cell counting. The results are shown in the appendix. Figure 3 .

[0229] The results showed that the cell amplification fold under the scheme of Example 3 of the present invention reached more than 500 times, while the amplification fold of Comparative Case 1 was only 100 times. This indicates that CD34+ cells can be induced into high-purity γδT cells through a pure factor scheme. It also shows that the addition of recombinant human DLL4 protein as Notch signaling agonist and the addition of TGF-β1 factor to the induction culture medium of the present invention have better induction differentiation performance of CD34+ cells.

[0230] In summary, the in vitro induction and culture method for umbilical cord blood-derived γδT cells described in this invention obtains a large number of hematopoietic stem cells through in vitro sorting and expansion of CD34+ cells, followed by induction differentiation into γδT cells using a suitable induction culture medium, and then proliferation culture using a suitable in vitro proliferation culture medium. This method not only achieves efficient expansion of umbilical cord blood-derived γδT cells at relatively low concentrations, significantly improving the expansion capacity of γδT cells and effectively solving the problem of insufficient expansion of umbilical cord blood γδT cells, but also, by using pure factors for expansion, effectively avoids interference from trophoblast cells, ensuring the safety of cell reinfusion and providing a more reliable solution for clinical applications, thus meeting clinical needs.

[0231] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. An in vitro induction medium for umbilical cord blood-derived γδ T cells, characterized by, An in vitro induction composition comprising a basal medium and umbilical cord blood-derived γδ T cells; the in vitro induction composition consists of: IL-7, a differentiation inducer, a Notch signal agonist, and plasma, wherein, the concentration of the IL-7 is 5-15 ng / mL based on the basal medium; the differentiation inducer is TGF-β1, and the concentration of the TGF-β1 is 1-1.5 ng / mL based on the basal medium; the Notch signal agonist is a recombinant human DLL4 protein, and the concentration of the recombinant human DLL4 protein is 1.5-2.5 μg / mL based on the basal medium; and the concentration of the plasma is 8-15 % v / v based on the volume of the basal medium.

2. An in vitro induction culture method of umbilical cord blood-derived γδ T cells, characterized by, comprising the following steps: (1) sorting CD34+ cells based on umbilical cord blood and performing seed cell culture to obtain CD34+ hematopoietic stem cells; (2) introducing the CD34+ hematopoietic stem cells into the in vitro induction medium of the umbilical cord blood-derived γδ T cells of claim 1 for induction culture; (3) continuing to supplement the in vitro expansion medium for expansion culture, thereby obtaining the desired γδ T cells; wherein in the step (3): the in vitro expansion medium comprises the following components at the following concentrations: IL-2 150-250 U / mL, IL-15 20-30 ng / mL, zoledronic acid 3-8 μM / mL, and plasma with a final concentration of 3-8 % v / v.

3. The in vitro induction and culture method for umbilical cord blood-derived γδT cells according to claim 2, characterized in that, In the step (1), the step of sorting CD34+ cells based on umbilical cord blood comprises: S1, extracting umbilical cord blood mononuclear cells, aspirating the white membrane layer, and adding buffer for resuspension to obtain a cell suspension; S2, diluting the cell suspension to a cell concentration of 1 x 10 8 -5 x 10 8 cells / mL; S3, incubating the antibody mixture reagent of the diluted cell suspension to obtain a sorting system; S4, repeatedly sorting the sorting system using a flow cytometer and magnetic beads, thereby obtaining sorted CD34+ hematopoietic progenitor cells.

4. The method of claim 2 or 3, wherein the umbilical cord blood-derived γδ T cells are induced in vitro by culturing the umbilical cord blood-derived γδ T cells in the presence of a cytokine. In the step (1), the temperature of the seed cell culture step is 35-38°C, and the culture period is 5-8 days; the seed cell culture medium of the seed cell culture step comprises a stem cell serum-free medium and a hematopoietic stem cell supplement; wherein, the stem cell serum-free medium accounts for 80-90 wt% of the total amount of the seed cell culture medium; the hematopoietic stem cell supplement accounts for 10-20 wt% of the total amount of the seed cell culture medium.

5. The in vitro induction and culture method for umbilical cord blood-derived γδT cells according to claim 2, characterized in that, In the step (2), the conditions of the induction culture step comprise: a culture temperature of 35-38°C; a culture condition of culturing under 5% v / v CO2; and a culture time of 2-3 days.

6. The method for inducing and culturing umbilical cord blood-derived γδ T cells in vitro according to claim 2, wherein the step of culturing the umbilical cord blood-derived γδ T cells in the presence of the cytokine is performed in the presence of IL-2 and IL-15. In the step (3): the conditions of the expansion culture step comprise: a culture temperature of 35-38°C; a culture condition of culturing under 5% v / v CO2; and a culture time of 10-15 days.

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