Application of Dynamically Cross-linked Hydrogels for Cell Culture

Abstract

The present invention relates to the application of a dynamically cross-linked hydrogel. Specifically, the dynamically cross-linked hydrogel is formed by mixing gelatin and a cross-linking group-modified cyclodextrin into a hydrogel prepolymer. After embryonic stem cells or immune cells are mixed in, a cross-linking reaction occurs under the action of a cross-linking initiator or ultraviolet light to produce the dynamically cross-linked hydrogel. The hydrogel maintains the stemness of embryonic stem cells in a basal culture medium without the need for feeder cells or differentiation inhibitors, is simple to operate, and is suitable for large-scale culture. The immune cell-loaded hydrogel can be directly used for injection and filling in vivo.

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering technology, and more particularly to the application of a dynamic cross-linked hydrogel in 3D cultured cells. Background Art

[0002] Since cell clusters are widely used in in vitro models for drug screening, basic research on disease progression and developmental biology, and clinical research on autologous or allogeneic organ cultivation and tissue regeneration, etc., the use of hydrogels for proliferation and culture of cell clusters, cytological research, cell release and collection, and as tissue engineering scaffolds for loading cells for tissue regeneration has important biomedical significance and application prospects.

[0003] In vitro culturing of cells, for those that grow in clusters, inhibiting differentiation during the culture process remains a crucial issue. For example, in the proliferation and culture of embryonic stem cells, mouse embryonic fibroblasts (MEFs) that have been irradiated with ultraviolet light to stop dividing can be used as feeder cells to inhibit differentiation. Embryonic stem cells (ESCs) are a typical cell line that grows in clusters. Generally speaking, conventional two-dimensional (2D) culture of ESCs requires co-culture with mouse embryonic fibroblasts (MEFs), or feeder cells, or culture medium supplements such as GSK3β and Mek1 / 2 inhibitors (2i) to inhibit spontaneous differentiation. However, maintaining ESCs in an undifferentiated state during long-term culture remains challenging. More importantly, unlike ESCs cultured on two-dimensional surfaces in vitro, during embryonic development, cells within the inner cell cluster interact with a three-dimensional (3D) extracellular matrix (ECM), which regulates the self-renewal and differentiation of ESCs. The interaction between cells and the surrounding ECM is crucial for ESC proliferation, stemness maintenance, directed differentiation, and organ formation. However, in the above-mentioned two-dimensional cell culture methods, whether using feeder layer cells or adding differentiation inhibitors, maintaining the undifferentiated state during the culture process can only last for a few days, and there is no method that can support the long-term growth of cell clusters.

[0004] Three-dimensional biomaterial scaffolds, such as hydrogels, are expected to assist in the long-term culture of embryonic stem cells without a feeder layer and without the need for frequent subculture. Although there have been a large number of studies on the application of hydrogel systems in in vitro three-dimensional cell culture, these works still have some problems to be solved. Existing methods for culturing embryonic stem cells in vitro cannot obtain large, uniformly sized clusters of cell clones, and it is difficult to maintain the stemness of embryonic stem cells during long-term culture. Feng Qian et al. reported the application of a gelatin-based host-guest supramolecular assembly hydrogel in the culture of mesenchymal stem cells. Within 14 days, mesenchymal stem cells differentiated into stellate osteocytes and could be injected into the joints of mice. However, there are no reports that this hydrogel can be used to culture embryonic stem cells in three dimensions for a long time while maintaining their stemness.

[0005] With the rapid development of immunotherapy, in vitro culture of immune cells has gained increasing attention among researchers. Traditional immune cell culture methods utilize two-dimensional or suspension cultures. To achieve optimal cell proliferation, various auxiliary reagents, such as differentiation inhibitors, are often added. Suspension cultures often require additives to prevent cell sedimentation. Some culture methods produce uneven cell growth, which is significantly affected by the growth environment and experimental procedures, making them unsuitable for large-scale culture. Summary of the Invention

[0006] One of the objectives of this application is to provide a gelatin-based dynamically cross-linked hydrogel for use in in vitro cell culture. The dynamically cross-linked hydrogel is a hydrogel prepolymer formed by mixing gelatin and a cyclodextrin modified with a cross-linking group, which undergoes a cross-linking reaction under a cross-linking initiator or light; wherein the cells are embryonic stem cells or immune cells.

[0007] In a preferred embodiment, the cross-linking group-modified cyclodextrin is acrylated cyclodextrin. In certain embodiments, the acrylated cyclodextrin is β-cyclodextrin.

[0008] In one embodiment, the method for preparing the hydrogel comprises the following steps:

[0009] 1) Preparation steps of acrylated β-cyclodextrin:

[0010] β-cyclodextrin is dissolved in dimethylformamide (DMF) to obtain a 1-10% (w / v) β-cyclodextrin / DMF solution, triethylamine is added at a ratio of 0.75-7.5% (v / v), and the system temperature is lowered to 0°C; acryloyl chloride is added dropwise in an amount 4-8 times the molar amount of cyclodextrin to the system, and stirring is continued for 12 hours after the addition is completed. Triethylamine hydrochloride is removed by suction filtration to obtain a clear solution, i.e., a reaction product; the reaction product is concentrated by vacuum rotary evaporation, the concentrated reaction product is added dropwise to acetone to obtain a white precipitate, the obtained white precipitate is washed with acetone, and finally, vacuum dried to obtain acrylated β-cyclodextrin (Ac-CD);

[0011] 2) Preparation steps of host-guest gelatin-cyclodextrin hydrogel:

[0012] Mixing gelatin and acrylated β-cyclodextrin and dissolving them in PBS to obtain a hydrogel prepolymer, wherein the gelatin solid content is 1-20% (w / v) and the Ac-CD solid content is 1-20% (w / v); preferably, the gelatin solid content is 4-10% (w / v) and the Ac-CD solid content is 5-15% (w / v);

[0013] 3) Adding a photoinitiator to the hydrogel prepolymer solution and irradiating it with ultraviolet light to obtain a degradable dynamic hydrogel (GelCD).

[0014] Preferably, the method for preparing the hydrogel comprises the following steps:

[0015] 1) Preparation steps of acrylated β-cyclodextrin:

[0016] β-cyclodextrin was dissolved in dimethylformamide (DMF) to obtain a 6% (w / v) β-cyclodextrin / DMF solution, triethylamine was added at a ratio of 4.5% (v / v), and the system temperature was lowered to 0°C;

[0017] Acryloyl chloride (7 times the molar amount of cyclodextrin) was added dropwise to the system. After the addition was completed, stirring was continued for 12 hours. Triethylamine hydrochloride was removed by filtration to obtain a clear solution, namely the reaction product. The product concentrate was added dropwise to 10 times the volume of acetone to obtain a white precipitate. The obtained white precipitate was washed with acetone and finally dried in vacuum to obtain acrylated β-cyclodextrin (Ac-β-CD).

[0018] 2) Preparation steps of host-guest gelatin-cyclodextrin hydrogel:

[0019] Gelatin and acrylated β-cyclodextrin are mixed and dissolved in PBS to obtain a hydrogel prepolymer. As a preferred technical solution of the present invention, the gelatin solid content in the hydrogel prepolymer is 8% (w / v) and the Ac-CD solid content is 10% (w / v).

[0020] 3) Adding I 2959 photoinitiator to the hydrogel prepolymer solution, mixing in cells, and obtaining a cell-loaded dynamically cross-linked hydrogel under 365 nm ultraviolet light initiation conditions. The hydrogel is added to the culture medium and cultured statically in an incubator.

[0021] The hydrogel of the present invention is formed based on host-guest interactions. The host-guest recognition reaction has the characteristics of mild reaction conditions and a dynamically reversible reaction process. It can not only effectively overcome the limitations of covalent binding, but also simulate biological functions at the molecular level. The host-guest interaction is based on non-covalent bonds. Non-covalent interactions include van der Waals forces, electrostatic attraction, hydrophobic interactions, and hydrogen bonds, which are key to generating host-guest recognition. The binding constant between host and guest molecules can be adjusted according to different guest molecular structures. Therefore, the dynamic cross-linked hydrogel of the present invention has a high degree of dynamic controllable performance.

[0022] In a preferred embodiment, in the hydrogel prepolymer of the present invention, the gelatin solid content is 4-10% (w / v), and the Ac-CD solid content is 5-15% (w / v); in certain embodiments, the ratio of the acrylate groups grafted on the cyclodextrin to the cyclodextrin is 0.8-1.8.

[0023] Preferably, the initiators used in this application include: visible light initiators, such as phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and phenyl (2,4,6-trimethylbenzoyl) phosphate sodium salt, which can perform cross-linking reaction in the blue light range (wavelength 405nm);

[0024] UV photoinitiators, such as 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (I2959), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (I819), benzil bismethyl ether (I651), α-ketoglutaric acid, etc., initiate crosslinking reaction under ultraviolet light range (320nm-400nm);

[0025] Infrared photoinitiators, such as bacteriochlorophyll a, polymethines, dye-borate, and alkyne cyanine dye 718, initiate cross-linking reactions under irradiation in the infrared range.

[0026] The dynamic cross-linked hydrogel can be rapidly degraded, thereby enabling the recovery and culture of embryonic stem cells. The degradation method can be to add collagenase, preferably at a concentration of 0.03%-0.3% (w / v); this method can enable the dynamic cross-linked hydrogel to achieve rapid degradation within 15 minutes. Another degradation method is to add guest small molecules with a host-guest binding constant with cyclodextrin greater than that of gelatin, such as adamantane, tert-butylbenzene, ibuprofen, geraniol, menthol, oleic acid, etc.; the amount added is 0.01%-0.1% (w / v). This degradation method is simple to operate and achieves degradation of the dynamic cross-linked hydrogel within minutes, achieving efficient and rapid recovery of cells; in addition, the method of recovering cells by adding guest small molecules will not affect the structure of cell clusters due to the addition of enzyme-active components such as collagenase.

[0027] Another object of the present application is to provide a method for culturing embryonic stem cells using the dynamic cross-linked hydrogel, comprising the following steps:

[0028] Gelatin and acrylated β-cyclodextrin were mixed and dissolved in PBS to obtain a hydrogel prepolymer. As a preferred technical solution of the present invention, the hydrogel prepolymer has a gelatin solid content of 8% (w / v) and an Ac-CD solid content of 10% (w / v);

[0029] A photoinitiator is added to a hydrogel prepolymer solution, and embryonic stem cells are mixed therein. Under light-induced conditions, a degradable dynamic hydrogel loaded with cells is obtained, and a culture medium is added thereto, and the hydrogel is statically cultured in an incubator.

[0030] The loaded cells are recovered every 7 days by adding collagenase or guest small molecules and loaded into freshly prepared dynamic cross-linked hydrogels for culture. In one embodiment, the culture medium comprises Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 1 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM NEAA, 50 units of penicillin / streptomycin, 0.1 mM b-mercaptoethanol, and 1000 units / ml of leukemia inhibitory factor (LIF).

[0031] Another object of the present application is to provide a method for culturing pluripotent embryonic stem cell clusters in vitro, the method comprising loading the embryonic stem cells into the dynamically cross-linked gelatin hydrogel and culturing them in a basal medium. Preferably, the basal medium is selected from one of calcium (Ca)-free MEM medium, basal Eagle medium (BME), F12, RPMI 1640, DMEM, MEM / F12, and DEME / F12 medium.

[0032] The gelatin-based dynamically cross-linked hydrogels described herein can be used to culture embryonic stem cells (ESCs) in a basal medium for long periods in vitro, while maintaining the clonal, undifferentiated cluster structure of the ESCs and the pluripotency of the ESCs without the use of feeder cells or differentiation inhibitors. In one embodiment, the ESCs maintained an undifferentiated, spherical cluster structure and possessed the pluripotency of all three germ layers after 20 days of culture in the dynamically cross-linked hydrogels.

[0033] In one embodiment, mESCs were cultured in GelCD hydrogels for up to 2 months to evaluate the effectiveness of GelCD hydrogels in maintaining the multipotency of loaded mESCs during long-term 3D culture. During the 2-month culture process, cells were passaged every 7 days, removed from the GelCD hydrogel using collagenase and re-loaded in freshly prepared GelCD hydrogels. After 9 cell passages, mESC colonies maintained a typical undifferentiated spherical morphology in the GelCD hydrogel. mESCs cultured for a long time in GelCD hydrogels were then recovered and subsequently seeded on MEFs. mESCs recovered from GelCD hydrogels could still form tight cell clusters and had strong immunofluorescence staining for nuclear Nanog and Oct3 / 4, in the same manner as mESCs conventionally passaged on MEFs. Recovered mESCs were also cultured in suspension to evaluate the spontaneous differentiation of mESCs and the formation of embryonic bodies (EBs). Nestin-, α-fetoprotein-, and α-SMA-positive cells, representing differentiated cells from the three germ layers, could be found in the formed EBs, indicating that mESCs obtained after 2 months of 3D culture in GelCD hydrogels were functionally pluripotent. These findings collectively demonstrate that GelCD hydrogels are an effective 3D culture platform that supports the proliferation and self-renewal of mESCs in long-term culture without the need for feeder cells or 2i supplements in the culture medium.

[0034] In another embodiment, the dynamic cross-linked hydrogel is used to culture immune cells in 3D, and the immune cells cultured by the immune cell culture medium include one or more of T cells, NK cells, DC cells or CIK cells. After the dynamic cross-linked hydrogel is loaded with immune cells, the immune cells are cultured in a common culture medium supplemented with CD3 antibodies and CD28 antibodies. Compared with the traditional two-dimensional method or suspension culture method, the growth rate of immune cells cultured using dynamic cross-linked hydrogel is faster and uniform cell clusters are formed. The dynamic cross-linked hydrogel based on gelatin has injectable properties, and the cultured immune cells can be directly used for injection without recycling operation.

[0035] The hydrogel of the present invention is used for 3D culture of cell types that grow in clusters, such as embryonic stem cells and immune cells, and has been shown to provide the dynamic microenvironment required by these cells. On the one hand, the dynamically cross-linked hydrogel itself has good dynamic properties. While providing three-dimensional support for cells, its excellent elastic modulus promotes cell growth in clusters and maintains their activity. On the other hand, gelatin is rich in polypeptides that can have good adhesion to certain cells. For example, gelatin contains positively charged lysine and arginine residues, which have specific cell adhesion sites (RGD groups), thus promoting cell adhesion and better simulating the effects of the extracellular matrix. By culturing embryonic stem cells and immune cells under conditions that simulate the in vivo environment, the growth rate and activity of cells are significantly improved, and the resulting cell clusters are particularly uniform in size. This method is simple to operate, suitable for large-scale in vitro cell culture, and has strong industrial application prospects.

[0036] Another object of the present application is to provide an application of a dynamic cross-linked hydrogel in the preparation of an injectable preparation, wherein the cell-loaded dynamic cross-linked hydrogel is injected or filled into the area to be treated in the patient's body, wherein the cells can remain active in the body for a long time, and under the action of collagenase in the body, the dynamic cross-linked hydrogel is gradually degraded to release the loaded cells, thereby achieving precise drug delivery to the lesion site.

[0037] It is understood that the embryonic stem cells described in this application include non-human embryonic stem cells. Preferably, the embryonic stem cells are non-human embryonic stem cells.

[0038] The hydrogel of the present invention is based on the host-guest interaction of gelatin and cyclodextrin, and has injectable properties. The cultured cells can be directly injected into the part to be treated in the patient's body. Under the action of enzymes such as collagenase in the body, the hydrogel can be gradually degraded, releasing cells to achieve treatment or other functions. The cells loaded in the hydrogel can also maintain their activity in the in vivo environment, and have good application prospects for the immunotherapy of tumors. It is particularly suitable for injecting the hydrogel loaded with immune cells into the part after tumor resection, where the immune cells can remain active, thereby producing a continuous killing effect on the cancer cells remaining in the tumor resection site, thereby achieving better surgical results. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 (a) Schematic diagram of the dynamic host-guest crosslinking structure of GelCD hydrogel. (b) At 37°C, the dynamic GelCD hydrogel undergoes a sol-gel transition under alternating high (500%) and low (1%) shear strains.

[0040] Figure 2(a) Representative electron microscopy images of mouse embryonic stem cell clusters loaded into dynamic cross-linked hydrogels or cultured using traditional 2D culture methods ("2i") and "MEFs" on days 1, 2, 4, and 7. (b) Cell colony size of mouse embryonic stem cell clusters in dynamic cross-linked hydrogels, 2D culture methods ("2i"), and MEFs over a 7-day culture period.

[0041] Figure 3 (a) Immunostaining results for SOX2 and F-actin on day 7. (b) Quantification of stemness markers SOX2, Nanog, and Oct3 / 4 expression by qPCR in mouse stem cell clusters from GelCD, 2i, and MEF groups, n=3.

[0042] Figure 4 Results of immunostaining for differentiation markers associated with the three germ layers in embryoid bodies formed from embryonic stem cells obtained after 20 days of long-term culture in GelCD hydrogel.

[0043] Figure 5 Cell counts of CD8+ T cells after 5 days of proliferation.

[0044] Figure 6 LCK and ZAP 70 immunofluorescence staining experiments after 7 days of CD8+ T cell proliferation. DETAILED DESCRIPTION

[0045] Instruments and reagents

[0046] Laser confocal microscope (Nikon ECLIPSE TE2000-U )

[0047] Fluorescence microscope (LECIA THUNDER IMAGER LIVE CELL SYSTEM)

[0048] Embryonic stem cell source: Cryoport Systems, Inc.

[0049] Source of CD8+T cells: Shanghai Cell Bank, Chinese Academy of Sciences.

[0050] Other reagents were commercially available.

[0051] Example 1 Preparation of GelCD Hydrogel

[0052] 1) Preparation steps of acrylated β-cyclodextrin

[0053] Dissolve 10 g of β-cyclodextrin in 150 mL of dimethylformamide (DMF), add 7 mL of triethylamine, and lower the system temperature to 0°C;

[0054] In a fume hood, add 5 mL of acryloyl chloride dropwise to the system over a period of 1 hour to prevent excessive substitution. After the addition is complete, a large amount of white suspended matter will form in the solution, which is triethylamine hydrochloride.

[0055] The triethylamine hydrochloride is removed by suction filtration to obtain a clear solution, which is the reaction product;

[0056] The reaction product was concentrated by vacuum rotary evaporation to about 20-50 mL, precipitated in 800 mL of acetone, and the white precipitate was collected by filtration and washed 2-3 times with a large amount of acetone. It was then vacuum dried to obtain acrylated β-cyclodextrin with a degree of substitution of approximately 1.

[0057] 2) Preparation steps of host-guest gelatin-cyclodextrin hydrogel:

[0058] Gelatin and acrylated β-cyclodextrin were mixed and dissolved in PBS to obtain a GelCD hydrogel prepolymer, wherein the gelatin solid content in the hydrogel prepolymer was 8% (w / v) and the Ac-CD solid content was 10% (w / v).

[0059] 3) Adding I 2959 photoinitiator to the hydrogel prepolymer solution, and obtaining a dynamically cross-linked hydrogel GelCD under the condition of 365 nm ultraviolet light initiation.

[0060] Example 2 Dynamic Mechanical Properties of GelCD Hydrogel

[0061] like Figure 1 As shown in the schematic diagram a, Ac-β-CD is used as a host monomer to complex with gelatin aromatic residues (such as phenylalanine, tyrosine and tryptophan). Through the host-guest complexation of Ac-β-CD with gelatin, the precursor solution obtained is photopolymerized to generate gelatin-cyclodextrin host-guest hydrogel (GelCD hydrogel). The dynamic properties of GelCD hydrogel were further studied. Under alternating high shear strain (500%) and low shear strain (1%), GelCD hydrogel experienced a transition between "liquid state" and "gel state". When the hydrogel returned to the "gel" state, the G' value almost immediately recovered to the initial level ( Figure 1 b) In summary, the rapid relaxation, shear thinning, and self-healing behaviors of the GelCD hydrogel demonstrated its highly reversible and dynamically cross-linked structure. This network can readily rearrange itself under the mechanical forces of the microenvironment, thus significantly facilitating cell cluster expansion.

[0062] Example 3 GelCD hydrogel for 3D cell culture

[0063] Cell culture methods

[0064] Embryonic stem cells (mESCs) were mixed with GelCD hydrogel prepolymer at a density of 1.5 × 10 5 50ul of cell-loaded hydrogel prepolymer was added. Photoinitiator was added and irradiated with 365nm UV light for 10 minutes to obtain cell-loaded hydrogel. 500ul The cell-laden hydrogel was infused with culture medium, which was replaced daily. The basal medium consisted of Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 15% mESC-appropriate fetal bovine serum (FBS, Millipore), 1 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Gibco), 0.1 mM NEAA (Gibco), 50 units of penicillin / streptomycin (Hyclone), 0.1 mM β-mercaptoethanol (Gibco), and 1000 units / ml of leukemia inhibitory factor (LIF, Millipore).

[0065] Cell recovery method

[0066] After long-term culture, collagenase was added to the cell-loaded hydrogel to degrade the hydrogel and collect the cultured cell clusters.

[0067] Control experiment

[0068] The effectiveness of dynamic GelCD hydrogels in supporting the expansion of mESCs in 3D culture was evaluated by comparing two conventional 2D culture methods using MEF feeder cells and GSK3β and Mek 1 / 2 (2i) inhibitors.

[0069] For the MEF control group, mESCs were cultured in the above-described basal medium using inactivated mouse embryonic fibroblasts (MEFs, Merck) as feeder cells at 37°C and 5% CO2. The medium was changed daily, and mESCs were passaged onto fresh MEFs every two days using TrypLE Express enzyme (Gibco).

[0070] For the 2i culture method control group, the overall protocol was similar to that of the MEF method group, except that mESCs were cultured on gelatinized culture dishes (by incubating with 0.1% gelatin solution from Millipore for at least 1 hour) instead of MEFs. The culture medium was the above-mentioned basal medium supplemented with additional GSK3β and Mek 1 / 2 inhibitors (0.1 mM PD03259010 and 0.1 mM CHIR99021, i.e., 2i).

[0071] During the 7-day culture period, embryonic stem cells were cultured using the above method, and cell morphology was observed daily under an electron microscope. Growth of individual embryonic stem cell clusters was compared. The maximum cross-sectional area of ​​the cell clusters was used to assess the growth of mouse embryonic stem cell clusters in dynamic hydrogels, 2i 2D culture, and MEFs (n=10). These data demonstrate the advantages of dynamic hydrogels over traditional 2D culture.

[0072] During the first 4 days, although the average maximum cross-sectional area of ​​3D ESC clusters in GelCD hydrogels was smaller than that of 2D cell clusters obtained using MEF or 2i methods, the cell cluster sizes became comparable among the three groups on day 7 ( Figure 2 a and b). The cell clusters formed in GelCD hydrogels are three-dimensional spheroids and therefore should contain more cells than similar-sized mESC clusters formed in two-dimensional culture methods, because two-dimensional cell clusters have a relatively flat morphology. Three-dimensional embryonic stem cell clusters in GelCD hydrogels are spherical and dense, while embryonic stem cell clusters generated by traditional two-dimensional methods are irregular in shape ( Figure 2 a), which is a manifestation of greatly reduced dryness.

[0073] Immunostaining for SOX2 and actin (F-actin) was performed on day 7. The 3D mESCs clusters formed in GelCD hydrogels were spherical, compact, and had strong stemness, whereas the 2D clusters formed in 2D culture methods (2i or MEF) were spreading and had low stemness.

[0074] In an experiment of immunofluorescence staining for embryonic stem cell pluripotency on day 7, cell clusters in 2D culture showed a loose structure, and cells at the edge of the cell clusters showed spreading filamentous actin structures and reduced expression of the stemness marker SOX2, which are characteristics that indicate cell differentiation. In contrast, cell clusters in dynamic GelCD hydrogels maintained their spherical actin arrangement and still had high expression of SOX2 on day 7 ( Figure 3 a). We further examined the pluripotency of mESCs in different culture groups using real-time polymerase chain reaction (qPCR, n=3). qPCR results showed that on day 7, the expression levels of pluripotency markers Sox2, Nanog, and Oct3 / 4 in the GelCD group were significantly higher than those in the 2i and MEF groups ( Figure 3 b).

[0075] Example 4: Long-term culture of embryonic stem cells in GelCD hydrogel

[0076] To further evaluate the effectiveness of GelCD hydrogel in maintaining the multipotency of loaded mESCs during long-term 3D culture, the GelCD hydrogel loaded with mESCs was subcultured in culture medium for up to 60 days according to the 3D method of Example 3. After 60 days of culture, the mESCs colonies in the GelCD hydrogel maintained typical Undifferentiated spherical Cells were then recovered from the GelCD hydrogels by degrading the hydrogels with collagenase and subsequently seeded onto MEFs. mESCs recovered from the GelCD hydrogels still formed compact, well-defined cell clusters, and nuclear immunofluorescence staining for Nanog and Oct3 / 4 was robust, comparable to that of mESCs routinely passaged on MEFs.

[0077] In this experiment, the recovered mESCs were also cultured in suspension to evaluate the spontaneous differentiation of mESCs and the formation of embryonic bodies (EBs). Immunostaining of differentiation markers related to the three germ layers (Nestin: ectoderm, α-fetoprotein: mesoderm and αSMA: endoderm) was performed in the embryonic bodies formed by embryonic stem cells obtained after 60 days of long-term culture in GelCD hydrogel. Through immunostaining experiments, cells positive for the three markers Nestin, α-fetoprotein and α-SMA were found in the formed EBs, representing the differentiated cells of the three germ layers respectively, indicating that the mESCs obtained after 60 days of three-dimensional culture in GelCD hydrogel still have functional pluripotency ( Figure 5 These findings collectively demonstrate that gelatin-based host-guest dynamic hydrogels are an effective three-dimensional culture platform that can support the proliferation and self-renewal of mESCs in long-term culture.

[0078] Example 5 GelCD in vitro 3D culture of immune cells

[0079] CD8 + T cells were obtained from splenocytes of adult mouse spleens and sorted by MojoSort according to the manufacturer's instructions (biolegend). TM The CD8+ T cells were isolated using a mouse CD8+ T cell isolation kit. The CD8+ T cells were collected by centrifuge and loaded into a gelatin dynamic cross-linked hydrogel according to the method of Example 3.

[0080] 500 μl of hydrogel prepolymer mixed with CD8+ T cells (cell concentration of 1-2×10 6 / ml), irradiated with 365 nm ultraviolet light for 10 minutes, and added RPMI 1640 medium supplemented with 5 ug / ml mouse CD3, 5 ug / ml mouse CD28, L-glutamine, 1% penicillin-streptomycin, 10% FBS and 50 ng / mL IL-2.

[0081] The 24-well plate was placed in a cell culture incubator at 37°C containing 5% CO2. On the third day of culture, half the volume of fresh culture medium containing 100 ng / mL IL-2 was added to the cell-loaded hydrogel to support T cell expansion.

[0082] At the same time, the 2D method was used to culture CD8+ T cells in vitro as a control, and the method was as follows:

[0083] 1) Dilute mouse CD3 antibody to 5 μg / ml in sterile PBS. Add 400 μl of the diluted antibody to each well of a 24-well plate and coat overnight at 4°C.

[0084] 2) The culture medium used was RPMI1640 + 10% serum + 5ug / ml mouse CD28 + double antibody, and the CD8 + T cell concentration was adjusted to 1×10 6 / ml;

[0085] Take the coated plate, discard the coating solution, and wash with sterile PBS three times; add 500 μl of cell suspension to each well and culture in a cell culture incubator at 37°C containing 5% CO2.

[0086] Transmission electron microscopy was used to observe cell growth morphology daily. After 7 days, it was found that T cells formed cell clusters, and the size of the cell clones was larger than that of traditional 2D culture methods.

[0087] 0.3% collagenase was added to the hydrogel, and after dynamic hydrogel degradation, naive CD8+ T cells were obtained and stained with 0.1% CFSE and incubated at 37°C for 30 minutes. The stained T cells were then thoroughly washed with sterile PBS and resuspended in 100uL PBS. 50k stained T cells were fixed with 2% PFA and stored in flow cytometry fluid at 4°C as a non-proliferation control. To quantify the effect of the hydrogel on T cell proliferation, naive CD8+ T cells stained with 0.5M CFSE were cultured in hydrogels and 2D culture methods respectively. After 5 days of culture, the cultured CD8+ T cells were counted using a hemocytometer to estimate the overall proliferation fold. The average number of cells in the cell clusters in the images of proliferating T cells was calculated by dividing the total cell number by the number of observed T cell clusters.

[0088] Dynamically cross-linked hydrogels promote CD8+ T cell proliferation

[0089] The effect of dynamic cross-linked hydrogels on T cell proliferation was evaluated after 7 days of cell culture. Bright field images showed that more T cell clusters were formed in the dynamic cross-linked hydrogels. For gelatin dynamic cross-linked hydrogels, the average number of T cells in such clusters was 12, while conventional 2D culture obtained only about 4 cells. Immunofluorescence staining of cultured cells on the seventh day was performed to detect the expression of ZAP 70 and LCK proteins. Figure 6 As shown, immune T cells cultured in the dynamic composite cross-linked hydrogels differentiated and proliferated well on day 7, forming cell clusters. The expression of ZAP 70 and LCK proteins was significantly higher than that of the control, indicating that immune T cells cultured in the dynamic cross-linked gelatin 3D maintained strong immune activity. Immune cells cultured in the dynamic cross-linked hydrogels can be directly injected in vivo. The loaded immune cells are gradually degraded by collagenase in vivo, and the released immune cells have strong immune activity and can directly kill tumor cells in vivo, playing a significant therapeutic role.

[0090] The above specific embodiments are exemplary illustrations of the technical solutions of the present invention. It should be understood that their purpose is to describe the content of the invention and not to limit the scope of the present disclosure. The scope of protection of the present invention shall be based on the scope defined by the claims of this application. Within the scope of knowledge possessed by ordinary technicians in the relevant technical field, equivalent changes and substitutions of various embodiments can be made without departing from the purpose of the present invention, and all should be considered to be included in the scope of the present invention.

Claims

1. A method for culturing cells in three dimensions in vitro, comprising the following steps: 1) Gelatin and cross-linking group-modified cyclodextrin are mixed to form a hydrogel prepolymer, which is then mixed with cells. A cross-linking reaction is initiated under light conditions using a chemical cross-linking initiator or a photoinitiator to produce a cell-loaded dynamic cross-linked hydrogel. 2) culturing the cell-loaded dynamic cross-linked hydrogel in a basic cell culture medium; It is characterized by: The cells are non-human embryonic stem cells, and the basal culture medium contains DMEM supplemented with 15% fetal bovine serum, 1 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM NEAA, 50 units of penicillin / streptomycin, 0.1 mM β-mercaptoethanol, and 1000 units / ml of leukemia inhibitory factor.

2. The method according to claim 1, characterized in that After step 2), the method further includes adding one or more of collagenase, adamantane, tert-butylbenzene, ibuprofen, geraniol, menthol, and oleic acid to the cultured dynamic cross-linked hydrogel to degrade the dynamic cross-linked hydrogel and recover the cultured cell clusters.

3. The method according to claim 1, characterized in that The cell-loaded dynamic cross-linked hydrogel gradually degrades and releases the cells in an in vivo environment.

4. Use of a dynamically cross-linked hydrogel in preparing a therapeutic agent, characterized in that: The dynamically cross-linked hydrogel is a cell-loaded dynamically cross-linked hydrogel obtained according to the method of claim 1 .

5. The use according to claim 4, characterized in that The dynamically cross-linked hydrogel is also loaded with therapeutic drugs.

6. The use according to claim 4, characterized in that The medicine is an injectable medicine.

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