A tissue resuscitation enhancer and methods of use and applications thereof

Abstract

The application discloses a tissue resuscitation enhancer and a use method and application thereof in the technical field of cell culture, and provides the tissue resuscitation enhancer, which comprises transferrin, LATS kinase inhibitor NIBR-LTSi, TGF-beta receptor kinase inhibitor SB431542, Rock kinase inhibitor Thiazovivin, vitamin C phosphate and superoxide dismutase SOD. The tissue resuscitation enhancer has a good resuscitation enhancement effect on frozen tissues, and can effectively improve the survival rate and expansion capacity of H1 embryonic stem cells and SD astrocytes after being resuscitated after being frozen, and accelerate the functional recovery of tissues or cells after being frozen, so as to guarantee the stability and repeatability of experiments.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a tissue resuscitation enhancer and its method of use and application. Background Technology

[0002] Currently, tissue and cell cryopreservation technology has been widely applied in fields such as biobanks, regenerative medicine, organoid culture, and clinical pathology sample preservation. The two commonly used cryopreservation methods, slow freezing and vitrification, are mainly suitable for small tissue samples down to the millimeter level, such as ovarian cortical strips. Cryopreservation and thawing systems for larger tissue samples down to the centimeter level are still underdeveloped.

[0003] During the cryopreservation and thawing of large-volume tissues, uneven freezing and thawing rates, as well as insufficient penetration of cryoprotectants, make it difficult for cells in the central region of the tissue to be effectively protected, leading to a large number of cell deaths. Furthermore, tissues contain various cell types, each with significant differences in their tolerance to freeze-thaw stress and cryoprotectant permeability. This can result in the selective loss of precious and fragile cell subpopulations during thawing, severely impacting the survival rate, structural integrity, and functional reconstruction of the tissue after thawing.

[0004] Currently, the mainstream commercial resuscitation additives on the market include two representative products: one is RevitaCell™ from Gibco, a brand under Thermo Fisher Scientific. Its formula contains only a specific ROCK inhibitor combined with simple antioxidant and free radical scavenging small molecules, resulting in a more singular target: ① It only mildly scavenges intracellular free radicals, without targeted ferroptosis blocking or collagen matrix repair functions, and has almost no effect on improving deep tissue hypoxia and oxidative damage; ② It does not have anti-fibrotic or tissue regeneration regulatory pathways, and only provides short-term relief for single-cell dissociation damage, and cannot maintain the integrity of large tissue structures; ③ It has a narrow applicable cell spectrum, with limited improvement only in the resuscitation of human pluripotent stem cells, and a slight benefit in the resuscitation of astrocytes and solid tumor tissues. Furthermore, it cannot be used in combination with other ROCK inhibitors, which greatly limits its use.

[0005] The second type is domestically produced CEPT, which contains Chroman 1, Emricasan, Polyamines, and Trans-ISRIB. It only targets and inhibits ROCK-mediated cell contraction damage, Caspase apoptosis, and endoplasmic reticulum stress pathways, and has three major shortcomings: ① It does not contain iron homeostasis regulating components, so it cannot block the accumulation of free iron caused by hypoxia in frozen tissues, and cannot inhibit the ferroptosis pathway, making it prone to lipid peroxidation damage in large tissue masses; ② It does not contain TGF-β pathway inhibitors, so cells are prone to fibrotic stress after freeze-thaw damage, destroying the three-dimensional structure of tissues and cell proliferation potential; ③ It lacks LATS / YAP pathway activators, so it cannot drive autonomous regeneration and repair after tissue damage, resulting in long-term cell expansion and poor differentiation uniformity after thawing; at the same time, its ROCK inhibitor Chroman 1 has a weak effect on the stability of E-cadherin adhesion between cells, and cell clusters are prone to fragmentation during freeze-thaw, making it unsuitable for the thawing of tumor organoids and large solid tissue masses.

[0006] It can be seen that existing commercial resuscitation additives generally have single effects and narrow applicability, making it difficult to meet the resuscitation needs of multiple tissues, stem cells and nerve cells at the same time. In particular, in the cryopreservation and resuscitation of surgical margin tissues such as lung cancer, breast cancer, colorectal cancer and ovarian cancer, as well as H1 embryonic stem cells and SD astrocytes, the low cell viability after resuscitation leads to poor experimental stability and reproducibility, which limits the relevant basic research and clinical translational application. Summary of the Invention

[0007] The purpose of this invention is to provide a tissue resuscitation enhancer and its method of use and application, thereby addressing the technical deficiencies existing in the prior art.

[0008] The present invention achieves the above objectives through the following technical solutions: In a first aspect, the present invention provides a tissue resuscitation enhancer, wherein the final concentration of each component added to the basal culture medium is used as the measurement basis, and the formulation of the tissue resuscitation enhancer is as follows: Transferrin 1-50 μg / ml; LATS kinase inhibitor NIBR-LTSi 0.3125-10 μM; TGF-β receptor kinase inhibitor SB431542 0.2-10 μM; Rock kinase inhibitor Thiazovivin 0.1-5 μM; Vitamin C phosphate L-ascorbate 2-phosphate 31.25-1000μM; Superoxide dismutase (SOD) 0.09375-0.375 KU / ml.

[0009] As a further optimization of the present invention, the formulation of the tissue resuscitation enhancer is based on the final concentration of each component added to the basal culture medium as the measurement standard: Transferrin 10-50 μg / ml; LATS kinase inhibitor NIBR-LTSi 2.5-10 μM; TGF-β receptor kinase inhibitor SB431542 0.2-0.5 μM; Rock kinase inhibitor Thiazovivin 0.1-0.5 μM; Vitamin C phosphate L-ascorbate 2-phosphate 125-500μM; Superoxide dismutase (SOD) 0.09375-0.375 KU / ml.

[0010] A second aspect of the present invention provides a method for using a tissue resuscitation enhancer in the resuscitation of cells or tissues for non-therapeutic purposes, comprising the following steps: (1) Thaw the frozen cells or tissues until a small amount of ice crystals remain. The specific operation method is based on the recommended recovery operation procedure for the cells or tissues. Further, thaw the frozen cells or tissues quickly in a 37°C water bath until only a small amount of ice crystals remain. (2) Add the tissue recovery enhancer described in any of the above items to the basic culture medium of the corresponding cultured cells or tissues to obtain a basic culture medium containing the tissue recovery enhancer; (3) Place the cells or tissues in a basal medium containing the tissue resuscitation enhancer and then replace it with a medium without the enhancer for further culture.

[0011] As a further optimization of the present invention, when applied to the resuscitation of H1 embryonic stem cells, the cells are cultured in a basal medium containing the tissue resuscitation enhancer for 1 day. If the cell condition is poor, the culture time after adding the resuscitation enhancer can be extended, but not exceeding 3 days. The medium is then partially replaced for 1 day, followed by a complete medium replacement and normal culture.

[0012] As a further optimization of the present invention, when applied to the resuscitation of SD astrocytes, the cells are cultured in a basal medium containing the tissue resuscitation enhancer for 3 days, cultured with half medium for 1 day, and then cultured normally with a complete medium change.

[0013] As a further optimization of the present invention, when applied to tissue resuscitation, the tissue-separated cells are mixed with matrix gel, dispensed, and solidified, and then cultured in a basic organoid culture medium containing the tissue resuscitation enhancer. After passage, the culture medium is replaced with a basic organoid culture medium without the tissue resuscitation enhancer. Furthermore, by continuously observing the size of the tissue-separated cells, passage is performed after the passage requirements are met.

[0014] A third aspect of the present invention provides the use of the tissue resuscitation enhancer as described in any one of (1)-(2) in any one of the above: (1) Preparation of cryopreserved tissue resuscitation reagent; (2) Preparation of cryopreserved cell resuscitation reagent.

[0015] As a further optimization of the present invention, the cryopreserved tissue includes the surgical margin tissue of any one of lung cancer, breast cancer, colorectal cancer, or ovarian cancer.

[0016] As a further optimization of the present invention, the cryopreserved cells include either H1 embryonic stem cells or SD astrocytes.

[0017] The beneficial effects of this invention are as follows: The tissue resuscitation enhancer provided by this invention comprises transferrin, LATS kinase inhibitor NIBR-LTSi, TGF-β receptor kinase inhibitor SB431542, Rock kinase inhibitor Thiazovin, and L-ascorbate. 2-phosphate and superoxide dismutase (SOD) form a novel iron homeostasis-enzymatic antioxidant closed loop, compensating for the lack of ferroptosis inhibition pathways in CEPT and specifically addressing the core pain points of large tissue layers with hypoxia and internal free iron bursts of oxidative damage. Thiazovin combines ROCK inhibition and E-cadherin stabilization functions, superior to Chroman1 in CEPT, reducing freeze-thaw fragmentation at the cell junction level. Combined with VC phosphate for continuous collagen synthesis to repair ECM, it fully preserves the three-dimensional tissue structure. Through two pathways completely absent in CEPT, SB431542 (TGF-β inhibition) and NIBR-LTSi (YAP regeneration activation), it simultaneously inhibits injury stress fibrosis and drives tissue self-repair, not only improving recovery survival rate but also enhancing long-term proliferation and differentiation uniformity after recovery. With multi-target synergistic coverage of six major tissue-specific recovery defects—oxidative damage, ferroptosis, mechanical fragmentation, fibrosis, insufficient regeneration, and cell contraction and apoptosis—its protective dimensions and long-term culture stability are significantly superior to CEPT, which only targets apoptosis and basal stress.

[0018] The tissue resuscitation enhancer provided by this invention has been confirmed by multiple parallel controlled trials to significantly improve cell viability in tumor margin tissues of colorectal cancer, lung cancer, breast cancer, and ovarian cancer compared to the control group, compared to GibcoRevitaCell™ and Yuanpei CEPT. For H1 embryonic stem cells, it can stably maintain stem cell stemness and reduce the proportion of spontaneous differentiation after resuscitation. For SD astrocyte primary cells, it can continuously retain the expression of GFAP characteristic markers, accelerating the functional recovery of tissues or cells after cryopreservation, thereby ensuring the stability and reproducibility of experiments. Attached Figure Description

[0019] Figure 1 The effect of adding different concentrations of transferrin alone on the proliferative capacity of colorectal cancer organoids provided by this invention; Figure 2 The effect of adding different concentrations of the LATS kinase inhibitor NIBR-LTSI alone on the proliferative capacity of colorectal cancer organoids provided by this invention; Figure 3 The effect of adding different concentrations of the TGF-β receptor kinase inhibitor SB431542 alone on the proliferative capacity of colorectal cancer organoids provided by this invention; Figure 4 The effect of adding different concentrations of the Rock kinase inhibitor Thiazovin alone on the proliferative capacity of colorectal cancer organoids provided by this invention; Figure 5 The effect of adding different concentrations of vitamin C phosphate L-ascorbate 2-phosphate alone on the proliferative capacity of colorectal cancer organoids provided by this invention; Figure 6 The effect of adding superoxide dismutase (SOD) alone on the proliferative capacity of colorectal cancer organoids provided by this invention; Figures 7-8 The effects of adding different concentrations of superoxide dismutase (SOD) and the optimal combination of five factors provided by this invention on the proliferation capacity of colorectal cancer organoids are shown in the figure. The blank group is the blank culture medium group without any added components. Figure 9 The figure shows the effects of the optimal concentration combination of four factors (NIBR-LTSI+SB431542+Thiazovivin+L-ascorbate 2-phosphate) and the optimal concentration combination of three factors (L-ascorbate 2-phosphate+SB431542+Thiazovivin) on the proliferation ability of colorectal cancer organoids. The blank group is the blank culture medium group without any added components, and the enhancement group is the blank culture medium with SOD and the optimal concentration combination of five factors added. Figure 10 The application effect of the tissue resuscitation enhancer provided by this invention on colorectal cancer resection margin tissue compared with competing products; Figure 11 The application effect of the tissue resuscitation enhancer provided by this invention on lung cancer resection margin tissue compared with competing products; Figure 12 The application test results of the tissue resuscitation enhancer provided by this invention and competing products on breast cancer surgical margin tissue; Figure 13The application effect of the tissue resuscitation enhancer provided by this invention on ovarian cancer resection margin tissue compared with competing products; Figure 14 The application effect of the tissue resuscitation enhancer provided by the present invention on H1 embryonic stem cells was tested. Figure 15 The effect of the tissue resuscitation enhancer provided by this invention on rat SD astrocytes was tested. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] Example 1: Investigation into the composition and effective concentration range of tissue resuscitation enhancers Different concentrations and types of supplementary factors (recovery enhancers) were added to the colorectal cancer organoid culture medium (Zhongke PRS-ICMA-3D) to prepare experimental culture media. The supplementary factors included transferrin, LATS kinase inhibitor NIBR-LTSi, TGF-β receptor kinase inhibitor SB431542, Rock kinase inhibitor Thiazovin, vitamin C phosphate L-ascorbate 2-phosphate, and superoxide dismutase SOD. The specific components and concentration designs are shown in Table 1. The concentration design was based on 1× tissue recovery enhancer.

[0022] Table 1. Design of components and concentrations in experimental culture media

[0023] Note: The 5-factor combination refers to the optimal concentration combination of five added factors: transferrin, NIBR-LTSi, SB431542, Thiazovivin, and L-ascorbate 2-phosphate (i.e., transferrin at 20 μg / ml, NIBR-LTSi at 5 μM, SB431542 at 0.2 μM, Thiazovivin at 0.5 μM, and L-ascorbate 2-phosphate at 250 μM).

[0024] The experimental culture media containing different concentrations and types of additives designed in Table 1 were added at a volume of 150 μL / well to a 96-well plate containing colorectal cancer organoid culture medium (Zhongke PRS-ICMA-3D). Each culture medium formulation was tested in 2 replicates. Cells obtained from the thawed and revived colorectal cancer resection margin tissue were isolated at 8×10⁻⁶ cells / mL.4 The cells are inoculated into a 96-well culture plate at a cell density of cells per well, and cultured at 37°C, 5% CO2, and 2% oxygen in culture medium formulations supplemented with different concentrations and different types of additive factors, respectively. The basal colorectal cancer organoid culture medium without the additive factors listed in Table 1 is used as the experimental control group.

[0025] After the start of culture, the culture medium is replaced every 3 days, and the culture is terminated when the size of the organoids exceeds 100 μm. After the culture is completed, the cell state is first recorded by bright-field photography under a microscope, and then the CellTiter-Glo (CTG) reagent is used to measure the intracellular adenosine triphosphate (ATP) content to evaluate cell viability. Within the same culture time, a higher CTG value indicates a higher cell viability and faster proliferation.

[0026] Unless otherwise specified, ordinary one-way ANOVA is used for statistical analysis of multiple sets of data in CTG data analysis, where P>0.05 is marked as ns, and 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0027] The CTG detection results of each additive factor are sequentially shown in Figure 1-6 , specifically: Figure 1 shows the effect of adding different concentrations of transferrin on the proliferation ability of colorectal cancer organoids. It can be seen from the figure that within the concentration range of 1-50 μg / ml, adding transferrin to the basal colorectal cancer organoid culture medium helps to improve cell viability and accelerate proliferation. It can be further seen from the figure that the preferred addition concentration of transferrin is 5.5-50 μg / ml.

[0028] Figure 2 shows the effect of adding different concentrations of NIBR-LTSI on the proliferation ability of colorectal cancer organoids. It can be seen from the figure that when the addition concentration of NIBR-LTSI exceeds 1.25 μM, the cell viability is significantly improved, while the improvement effect on cell viability is not obvious when the concentration is lower than 1.25 μM. It can be further seen from the figure that the preferred addition concentration of NIBR-LTSI is 2.5-10 μM.

[0029] Figure 3The figure shows the effect of adding different concentrations of SB431542 on the proliferation ability of colorectal cancer organoids. As can be seen from the figure, when the concentration of SB431542 exceeds 1 μM, it has an adverse effect on cell viability. Therefore, the preferred concentration of SB431542 is 0.2-0.5 μM.

[0030] Figure 4 The effect of adding different concentrations of Thiazovivin on the proliferation ability of colorectal cancer organoids is shown. The preferred concentration of Thiazovivin is 0.1-0.5 μM. Within this concentration range, the proliferation ability of colorectal cancer organoids is significantly improved.

[0031] Figure 5 The figure shows the effect of adding different concentrations of L-ascorbate 2-phosphate on the proliferation ability of colorectal cancer organoids. As can be seen from the figure, the preferred addition concentration of L-ascorbate 2-phosphate is 125-500 μM. Within this concentration range, the proliferation ability of colorectal cancer organoids is significantly improved.

[0032] In summary, the optimal concentrations for promoting proliferation of transferrin, NIBR-LTSi, SB431542, Thiazovivin, and L-ascorbate 2-phosphate in basic colorectal cancer organoid culture medium were 20 μg / ml, 5 μM, 0.2 μM, 0.5 μM, and 250 μM, respectively.

[0033] Figure 6 The study showed the effect of adding different concentrations of SOD alone on the proliferation capacity of colorectal cancer organoids. It was found that adding SOD alone to the basic colorectal cancer organoid culture medium did not have a significant effect on promoting proliferation.

[0034] Furthermore, different concentrations of SOD were combined with the optimal concentrations of the above-mentioned five additive factors and then added to the basic colorectal cancer organoid culture medium, such as... Figure 7-8 As shown, the combination of SOD with the above 5 added factors has a significant proliferative effect; the concentration of SOD with the best proliferative effect is 0.09375 KU / ml.

[0035] In summary, the optimal concentrations of the components in the 1× Tissue Resuscitation Enhancer formulation for promoting proliferation are as follows: transferrin 20 μg / ml, NIBR-LTSi 5 μM, SB431542 0.2 μM, Thiazovivin 0.5 μM, L-ascorbate 2-phosphate 250 μM, and SOD 0.09375 KU / ml.

[0036] Furthermore, the effects of the optimal concentration combinations of three added factors (L-ascorbate 2-phosphate + SB431542 + Thiazovivin) (denoted as the 3-factor group), the optimal concentration combinations of four added factors (NIBR-LTSI + SB431542 + Thiazovivin + L-ascorbate 2-phosphate) (denoted as the 4-factor group), the optimal concentration combinations of five added factors (transferrin + NIBR-LTSi + SB431542 + Thiazovivin and L-ascorbate 2-phosphate) (denoted as the 5-factor group), and 1× tissue resuscitation enhancer (enhanced group, transferrin 20 μg / ml, NIBR-LTSi 5 μM, SB431542 0.2 μM, Thiazovivin 0.5 μM, L-ascorbate 2-phosphate 250 μM, and SOD 0.09375 KU / ml) on the proliferative capacity of colorectal cancer organoids were compared.

[0037] The results are as follows Figure 9 As shown, the 1× tissue resuscitation enhancer (enhanced group) showed the best effect in promoting the proliferation of colorectal cancer organoids. The 1× tissue resuscitation enhancer (enhanced group) improved the colorectal cancer organoid proliferation capacity by approximately 71% compared to the factor 3 group, approximately 24% compared to the factor 4 group, and approximately 11% compared to the factor 5 group. CTG results were analyzed using ordinary one-way ANOVA, and the overall means of each group showed highly significant differences (P < 0.0001).

[0038] Example 2: Application Test of Tissue Resuscitation Enhancement Reagent According to the investigation in Example 1, the optimal concentrations of the components in the 1× tissue resuscitation enhancer formulation for promoting proliferation are as follows: transferrin 20 μg / ml, NIBR-LTSi 5 μM, SB431542 0.2 μM, Thiazovivin 0.5 μM, L-ascorbate 2-phosphate 250 μM, and SOD 0.09375 KU / ml.

[0039] 2.1. Experiments on the amplification and application of cut edge tissues An amplification experiment was conducted using 1× tissue resuscitation enhancer on resection margins of colorectal cancer, lung cancer, breast cancer, and ovarian cancer. Additionally, the efficacy was compared with that of Gibco-RevitaCell™ and CEPT (comprising Chroman 1, Emricasan, Polyamines, and Trans-ISRIB) sold by Yuanpei Company as competing products.

[0040] 2.1.1. Resection margin tissue of colorectal cancer (1) The frozen colorectal cancer resection margin tissue (from the company's sample bank) was quickly placed in a 37°C water bath to thaw until only a small amount of ice crystals remained; (2) Collect cells and count them according to the recommended operating procedure for the selected tissue type; (3) Following the recommended operating procedures for the cultured organoid type, the recovered 5E 4 -1E 6 Mix each cell with 1 mL of Matrigel and dispense the mixture into a cell culture plate. (4) Invert the culture plate and place it in a 5% CO2 incubator at 37°C for at least 30 minutes until the Matrigel matrix gel is completely solidified; (5) Add 1× tissue recovery enhancer to organoid culture medium for colorectal cancer organoids (Zhongke PRS-ICMA-3D), adding 100μL of 1× tissue recovery enhancer per 10ml of culture medium; (6) Slowly add organoid culture medium containing 1× resuscitation enhancer to each well along the well wall of the culture plate in step (4), and incubate in a 5% CO2, 37℃ incubator. Replace the organoid culture medium (containing 1× tissue resuscitation enhancer) according to the recommended operating procedure for the type of organoid being cultured. (7) The culture medium with added resuscitation enhancer should be changed every 3 to 4 days. The culture should be ended when the diameter of the cultured organoids is greater than 100 μm. After culture, the cell state was first recorded by bright-field microscopy, and then the cell viability was assessed by measuring the intracellular ATP (adenosine triphosphate) content using CTG (CellTiter-Glo) reagent.

[0041] The experimental groups are as follows: The organoid culture medium containing 1× tissue resuscitation enhancer was designated as the enhancement group; The control group consisted of organoid culture medium supplemented with an equal volume of PBS instead of 1× tissue resuscitation enhancer. Add a group of competitors of the same volume, where competitor 1 is Gibco-RevitaCell™; The enhancement group, blank group, and competitor 1 group each have three replicate samples (sample names are ZBOCP240536, ZBOCP240573, and ZBOCP241010, respectively).

[0042] For the CTG data of the three groups, ordinary one-way ANOVA was used for multi-group data statistics, in which P>0.05 is marked as ns, and 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0043] The results are shown in Figure 10 , the cell viability of the enhancement group is significantly better than that of the blank group and the competitor 1 group. It can be seen that the use of the tissue resuscitation enhancer can effectively improve the survival rate and expansion ability of the cells from cryopreserved colon cancer marginal tissues after resuscitation following cryopreservation.

[0044] With reference to the above experimental procedure, expansion application experiments were also carried out on lung cancer marginal tissues, breast cancer marginal tissues, and ovarian cancer marginal tissues.

[0045] 2.1.2 Lung cancer marginal tissues For lung cancer marginal tissues, two competitor groups were set up: the competitor 1 used in the competitor 1 group is Gibco-RevitaCell™; the competitor 2 used in the competitor 2 group is CEPT sold by Yuanpei Company; the blank group, enhancement group, competitor 1 group, and competitor 2 group each have three replicate samples (sample names are LUCP250224-N, LUCP250140-N, and LUCP250141-N, respectively).

[0046] For the CTG data of the four groups, ordinary one-way ANOVA was used for multi-group data statistics, in which P>0.05 is marked as ns, and 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0047] The results are shown in Figure 11 , the cell viability of the enhancement group is significantly better than that of the blank group, competitor 1 group and competitor 2 group. It can be seen that the use of the tissue resuscitation enhancer can effectively improve the survival rate and expansion ability of the cells from cryopreserved lung cancer marginal tissues after resuscitation following cryopreservation.

[0048] 2.1.3 Breast cancer marginal tissues For breast cancer margin tissues, two competitor groups were set up: Competitor 1 used in the Competitor 1 group was Gibco-RevitaCell™; Competitor 2 used in the Competitor 2 group was CEPT sold by Yuanpei Company; Three replicates of samples (sample names are BRCP250017, BRCP250017 and BRCP250034 respectively) were set for each of the blank group, enhancement group, Competitor 1 group and Competitor 2 group. Ordinary one-way ANOVA was used for multi-group data statistics of CTG data of the four groups, in which P>0.05 is marked as ns, 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0049] The results are shown in Figure 12 that the cell viability of the enhancement group is significantly better than that of the blank group, Competitor 1 group and Competitor 2 group. It can be seen that the use of the tissue resuscitation enhancer can effectively improve the survival rate and expansion ability of the cells in the frozen breast cancer margin tissues after resuscitation from cryopreservation.

[0050] 2.1.4 Ovarian cancer margin tissues For ovarian cancer margin tissues, two competitor groups were set up: Competitor 1 used in the Competitor 1 group was Gibco-RevitaCell™; Competitor 2 used in the Competitor 2 group was CEPT sold by Yuanpei Company; three replicates of samples (sample names are OVCP250148, OVCP250151 and OVCP240091 respectively) were set for each of the blank group, enhancement group, Competitor 1 group and Competitor 2 group. Ordinary one-way ANOVA was used for multi-group data statistics of CTG data of the four groups, in which P>0.05 is marked as ns, 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0051] The results are shown in Figure 13 that the cell viability of the enhancement group is significantly better than that of the blank group, Competitor 1 group and Competitor 2 group. It can be seen that the use of the tissue resuscitation enhancer can effectively improve the survival rate and expansion ability of the cells in the frozen ovarian cancer margin tissues after resuscitation from cryopreservation.

[0052] 2.2 H1 Embryonic Stem Cells (1) According to the recommended resuscitation procedure for the cultured H1 embryonic stem cells (Procell; CL-0889), quickly place the cryopreserved H1 embryonic stem cells in a 37°C water bath for thawing until only a small amount of ice crystals remain.

[0053] (2) Add 1× tissue resuscitation enhancer to the H1 embryonic stem cell medium (Procell; GCM-0889), add 100 μL of 1× tissue resuscitation enhancer per 10 mL of medium, culture in a 24-well plate, and the number of H1 embryonic stem cells per well is 1E 4 , the medium addition amount is 500 μL / well, and culture for 1 day. If the cell state is poor, the culture days after adding 1× tissue resuscitation enhancer can be extended, with a maximum of no more than 3 days; (3) Perform half medium change with H1 embryonic stem cell medium without 1× tissue resuscitation enhancer, culture for 1 day, then perform full medium change with H1 embryonic stem cell medium without tissue resuscitation enhancer, and carry out normal culture; The above group is recorded as the enhancement group, and a blank group is additionally set. The blank group uses H1 embryonic stem cell medium supplemented with an equal volume of PBS instead of 1× tissue resuscitation enhancer throughout the whole process; the competitor 1 used in the competitor 1 group is Gibco-RevitaCell™; the competitor 2 used in the competitor 2 group is CEPT sold by Yuanpei Company; the enhancement group and the blank group are each tested in 2 replicate wells; (4) When the confluence of H1 embryonic stem cells reaches 70-80%, the experiment can be terminated.

[0054] After the culture, first use a microscope to take bright-field photos to record the cell state, and then use CTG (CellTiter-Glo) reagent to measure the adenosine triphosphate (ATP) content in the cells to evaluate the cell viability.

[0055] The four groups of CTG data are analyzed by Ordinary one-way ANOVA for multi-group data statistics, in which P>0.05 is marked as ns, and 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0056] The results are shown Figure 14 as follows, the cell viability of the enhancement group is significantly better than that of the blank group. It can be seen that the use of the tissue resuscitation enhancer can effectively improve the survival rate and expansion ability of H1 embryonic stem cells after cryopreservation and resuscitation.

[0057] 2.3 SD rat astrocytes (1) Following the recommended resuscitation procedure for cultured rat SD astrocytes (Cyagen Biosciences; SCCAC-00001), the frozen rat SD astrocytes were quickly placed in a 37°C water bath to thaw until only a small amount of ice crystals remained.

[0058] (2) Add 1× tissue resuscitation enhancer to rat SD astrocyte culture medium, adding 100 μL of tissue resuscitation enhancer per 10 ml of culture medium, culture for 3 days, and culture in 24-well plates. The number of rat SD astrocytes per well is 1E. 4 The culture medium was added at a rate of 500 μL per well. (3) Replace half of the medium with SD astrocyte culture medium without 1× recovery enhancer, culture for 1 day, and finally replace the medium completely with SD astrocyte culture medium without 1× recovery enhancer and culture normally.

[0059] (4) The experiment can be ended when the confluence of rat SD astrocytes is 70%-80%.

[0060] After culture, the cell state was first recorded by immunofluorescence imaging using a fluorescence microscope, and the cell viability was assessed by measuring the intracellular ATP (adenosine triphosphate) content using CTG (CellTiter-Glo) reagent.

[0061] Further immunofluorescence assays were performed on SD astrocytes, with the following specific steps: (a) The rat astrocyte blank group consisted of an equal volume of PBS, while the enhancement group consisted of SD glial cell culture medium supplemented with 1× resuscitation enhancer. Competitor 1 used Gibco-RevitaCell™; competitor 2 used CEPT sold by Yuanpei Company. Cells were cultured until all 48 wells were confluent, and photographs were taken before fixation. (Three replicates were prepared; one replicate was used for immunofluorescence, and the other two replicates were used for microscopic examination and photographing, followed by direct addition of CTG to the wells and incubation at room temperature for 10 minutes. Data were plotted as bar charts for significance analysis.) (b) Aspirate the supernatant and wash the cells twice with PBS; (c) Fix cells with 4% paraformaldehyde for 15 minutes and wash cells three times with PBS; (d) Permeabilize cells with 0.3% Triton™ X-100 (diluted with PBS) at room temperature for 30 minutes, and wash cells three times with PBS; (e) Block cells with 5% BSA diluted in PBS at room temperature for 60 minutes; (f) Incubate the cells with the primary antibody (GFAP 1:800) diluted in 5% BSA (GFAP is the English abbreviation for glial fibrillary acidic protein, which is a marker of astrocyte activation, mainly distributed in astrocytes of the central nervous system) at 2-8°C overnight, then rinse the cells 3 times with PBS; (g) Incubate the cells with the secondary antibody (1:1000) diluted in 5% BSA for 60 minutes at room temperature, then rinse the cells 3 times with PBS; (h) Stain the cells with DAPI solution (3ng / mL) for 10 minutes; (i) Rinse the cells once with PBS; (j) Mount the slides with ProLong™ Gold Antifade Mountant; (k) Perform immunofluorescence imaging, and meet the data quality control standards.

[0062] Ordinary one-way ANOVA was used for statistical analysis of multiple groups of data in the four sets of CTG data, where P>0.05 is marked as ns, 0.01<P≤0.05 is marked , 0.001<P≤0.01 is marked , 0.0001<P≤0.001 is marked , P≤0.0001 is marked .

[0063] The results are shown in Figure 15 , the cell viability of the enhancement group is significantly better than that of the blank group. It can be seen that the use of the tissue resuscitation enhancer can effectively improve the survival rate and expansion ability of rat SD astrocytes after cryopreservation and resuscitation.

[0064] The above-mentioned examples only express several embodiments of the present invention, which are described relatively specifically and in detail, but should not be construed as a limitation on the patent scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and all of these shall fall within the protection scope of the present invention.

Claims

1. A tissue resuscitation enhancer, characterized in that, The formulation of the tissue resuscitation enhancer is as follows, based on the final concentration of each component added to the basal culture medium: Transferrin 1-50 μg / ml; LATS kinase inhibitor NIBR-LTSi 0.3125-10 μM; TGF-β receptor kinase inhibitor SB431542 0.2-10 μM; Rock kinase inhibitor Thiazovivin 0.1-5 μM; Vitamin C phosphate L-ascorbate 2-phosphate 31.25-1000μM; Superoxide dismutase (SOD) 0.09375-0.375 KU / ml.

2. The tissue resuscitation enhancer according to claim 1, characterized in that, The formulation of the tissue resuscitation enhancer is as follows, based on the final concentration of each component added to the basal culture medium: Transferrin 10-50 μg / ml; LATS kinase inhibitor NIBR-LTSi 2.5-10 μM; TGF-β receptor kinase inhibitor SB431542 0.2-0.5 μM; Rock kinase inhibitor Thiazovivin 0.1-0.5 μM; Vitamin C phosphate L-ascorbate 2-phosphate 125-500μM; Superoxide dismutase (SOD) 0.09375-0.375 KU / ml.

3. A method of using a tissue resuscitation enhancer in cell or tissue resuscitation for non-therapeutic purposes, characterized in that, Includes the following steps: (1) Thaw the frozen cells or tissues until a small amount of ice crystals remain; (2) Add the tissue recovery enhancer as described in any one of claims 1-2 to the basal culture medium of the corresponding cultured cells or tissues to obtain a basal culture medium containing the tissue recovery enhancer; (3) Place the cells or tissues in a basal medium containing the tissue resuscitation enhancer and then replace it with a medium without the enhancer for further culture.

4. The method of use according to claim 3, characterized in that, When applied to the resuscitation of H1 embryonic stem cells, the cells are cultured in basal medium containing the tissue resuscitation enhancer for 1 day, cultured with half medium replacement for 1 day, and then cultured normally with full medium replacement.

5. The method of use according to claim 3, characterized in that, When applied to the resuscitation of SD astrocytes, the cells are cultured in basal medium containing the tissue resuscitation enhancer for 3 days, then cultured with half medium for 1 day, and finally cultured normally with a complete medium change.

6. The method of use according to claim 3, characterized in that, When applied to tissue resuscitation, the isolated tissue cells are mixed with matrix gel, dispensed, and solidified. Then, they are added to a basic organoid culture medium containing the tissue resuscitation enhancer for culture. After passage, the culture medium is replaced with a basic organoid culture medium that does not contain the tissue resuscitation enhancer.

7. The application of a tissue resuscitation enhancer as described in any one of claims 1-2 in any one of (1)-(2), characterized in that: (1) Preparation of cryopreserved tissue resuscitation reagent; (2) Preparation of cryopreserved cell resuscitation reagent.

8. The application according to claim 7, characterized in that, The cryopreserved tissues include surgical margins from any one of the following cancers: lung cancer, breast cancer, colorectal cancer, or ovarian cancer.

9. The application according to claim 7, characterized in that, The cryopreserved cells include either H1 embryonic stem cells or SD astrocytes.

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