Environment-friendly cell preservation method

Abstract

The invention discloses an environment-friendly cell preservation method, and particularly relates to the field of cell preservation, the method comprises the following steps: step 1, cells to be preserved are placed in a pretreatment solution containing a natural saccharide protective agent (3-5% w / v) and an antioxidant (0.1-0.5% w / v) for incubation, the natural saccharide protective agent is trehalose (3-5% w / v), the antioxidant is a plant extracted polyphenol compound (0.1-0.5% w / v), and the pretreatment solution is mixed with the plant extracted polyphenol compound (0.1-0.5% w / v); the incubation temperature is 4-37 DEG C, and the incubation time is 30-60 minutes. Soybean protein isolate or pea protein is adopted to replace human serum albumin, ethical dispute is avoided, the degradation period is smaller than or equal to 30 days, the biodegradable buffer solution, PLGA and sodium alginate composite liquid are adopted to be completely degraded in the natural environment for 28 days, micro-plastic pollution is avoided, meanwhile, trehalose and plant polyphenol are adopted to replace DMSO, and the chemical toxicity risk is eliminated.

Description

Technical Field

[0001] This invention relates to the field of cell preservation, and more specifically, to an environmentally friendly cell preservation method. Background Technology

[0002] Traditional cell preservation methods mainly rely on liquid nitrogen freezing (-196°C) or chemical protectants (such as DMSO), which have significant drawbacks: Environmental pollution: DMSO metabolites are biotoxic, and plastic packaging such as cryopreservation tubes is difficult to degrade, leading to long-term accumulation and ecological burden. Ethical controversy: Animal-derived components such as human serum albumin need to be extracted from human plasma, which involves ethical controversy and is costly; Low preservation efficiency: There have been few breakthroughs in room temperature preservation technology, and existing methods typically result in cell viability of less than 50% (within 24 hours) at room temperature.

[0003] Existing improved technologies, such as CN116569917B, propose glycolysis inhibitor pretreatment combined with trehalose protection, but do not solve the problem of animal-derived components; CN114097771A uses heat shock protein to extend preservation time, but it is costly and depends on low temperature conditions; CN112640888B relies on ethanol fixation, which is only suitable for pathological samples and cannot preserve live cells.

[0004] There is an urgent need for an environmentally friendly, low-cost method that does not require cryopreservation and is suitable for the preservation of living cells, in order to address the environmental burden and operational limitations of traditional technologies. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution: an environmentally friendly cell preservation method, comprising the following steps: Step 1: Incubate the cells to be preserved in a pretreatment solution containing natural sugar protectants and antioxidants. The natural sugar protectant is trehalose (3-5% w / v), and the antioxidant is plant-extracted polyphenolic compounds (0.1-0.5% w / v). The incubation temperature is 4-37℃, and the time is 30-60 minutes. Step 2: Collect the pretreated cells by centrifugation, wash with phosphate-buffered saline (PBS), and resuspend in an environmentally friendly cell preservation solution; Step 3: Store the cell suspension at room temperature (18-25℃).

[0006] In a preferred embodiment, the environmentally friendly cell preservation solution comprises the following components: plant-derived protein substitute (10-30% w / v), low molecular weight heparin calcium (20-50 IU / ml), edaravone (0.1-1% w / v), glutamine (1-3 mM), and biodegradable buffer.

[0007] In a preferred embodiment, the pretreatment solution further includes the metabolic inhibitor 6-aminonicotinamide at a concentration of (20-50 nM).

[0008] In a preferred embodiment, the plant-derived protein substitute is soy protein isolate or pea protein, with a concentration of 15-25% w / v.

[0009] In a preferred embodiment, the biodegradable buffer solution is a PLGA and sodium alginate composite solution, where PLGA:sodium alginate = 3:1 and pH value is 7.0-7.4.

[0010] In a preferred embodiment, the preservation solution further includes heat shock protein (HSP70, 0.005-0.02% w / v).

[0011] The technical effects and advantages of this invention are as follows: This invention uses soy protein isolate or pea protein to replace human serum albumin, avoiding ethical controversies, and the degradation cycle is ≤30 days. It uses a biodegradable buffer solution, and the PLGA and sodium alginate composite solution is completely degraded in the natural environment in 28 days, avoiding microplastic pollution. At the same time, it uses trehalose and plant polyphenols to replace DMSO, eliminating the risk of chemical toxicity. Detailed Implementation

[0012] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0013] The features and performance of the present invention will be further described in detail below with reference to embodiments: Example 1: Validation of the effect of pretreatment solution containing metabolic inhibitors step: 1. Human umbilical cord mesenchymal stem cells (1×10^6 cells / mL) were placed in a pretreatment solution. The pretreatment solution consisted of: Group A: 5% trehalose + 0.3% tea polyphenols + 40 nM 6-aminonicotinamide; Group B: 5% trehalose + 0.3% tea polyphenols; Group C: No pretreatment (control); 2. Incubate at 4℃ for 30 minutes, centrifuge, wash with phosphate-buffered saline (PBS), and then add preservation solution. The preservation solution has the same composition for groups A, B, and C, all of which include 20% w / v soy protein isolate, 30 IU / ml low molecular weight heparin calcium, 0.4% w / v edaravone, 2 mM glutamine, and biodegradable buffer. The biodegradable buffer solution is a composite solution of PLGA and sodium alginate, with a ratio of PLGA:sodium alginate = 3:1 and a pH value of 7.3. 3. Store at 25℃ for 14 days and test for viability and metabolic activity (ATP content).

[0014] The obtained data is shown in Table 1 below:

[0015] As shown in Table 1, the addition of 6-aminonicotinamide can further inhibit metabolic consumption (slow down the decline of ATP) and significantly improve the survival rate (p<0.05). Example

[0016] step: 1. Pretreatment: Use 5% trehalose + 0.3% tea polyphenols + 40 nM 6-aminonicotinamide and incubate at 4℃ for 30 minutes; 2. Preservative solution composition: Group D: 20% soy protein isolate + low molecular weight heparin calcium 30 IU / ml, edaravone 0.4% w / v, glutamine 2 mM and biodegradable buffer; Group E: 20% pea protein + low molecular weight heparin calcium 30 IU / ml, edaravone 0.4% w / v, glutamine 2 mM and biodegradable buffer; Group F: 20% bovine serum albumin + low molecular weight heparin calcium 30 IU / ml, edaravone 0.4% w / v, glutamine 2 mM and biodegradable buffer; The biodegradable buffer solution is a composite solution of PLGA and sodium alginate, with a ratio of PLGA:sodium alginate = 3:1 and a pH value of 7.3. 3. Store at 25℃ for 14 days and test cell viability and morphological integrity.

[0017] The obtained data is shown in Table 2 below:

[0018] Based on the above, the bioavailability of plant-derived proteins is close to that of traditional albumin, and plant garden proteins are more easily degraded, with a degradation rate of >85%, making them significantly more environmentally friendly. Example

[0019] step: Pretreatment: Use 5% trehalose + 0.3% tea polyphenols + 40 nM 6-aminonicotinamide and incubate at 4℃ for 30 minutes; 2. Preservative solution composition: Group G: 20% soy protein isolate + low molecular weight heparin calcium 30 IU / ml, edaravone 0.4% w / v, glutamine 2 mM, biodegradable buffer and 0.01% HSP70 Group H: 20% soy protein isolate + low molecular weight heparin calcium 30 IU / ml, edaravone 0.4% w / v, glutamine 2 mM and biodegradable buffer; The biodegradable buffer solution is a PLGA and sodium alginate composite solution, with a PLGA:sodium alginate ratio of 3:1 and a pH of 7.3. 3. Store at 25℃ for 21 days (extend the storage time) and detect cell viability and apoptosis rate.

[0020] The obtained data is shown in Table 3 below:

[0021] As shown above, when the HSP70 concentration is ≥0.01%, the apoptosis rate decreases by >60%, significantly enhancing stress resistance. Example

[0022] Step 1: Prepare PLGA:sodium alginate complex buffer (3:1, 1:1, 1:3), pH 7.2; Step 2: Store CHO cells (1×10^8 cells / mL) at 25℃ and detect degradation cycle and cell adhesion rate.

[0023] The data obtained is shown in Table 4 below:

[0024] As can be seen from the above, the higher the PLGA content, the longer the degradation cycle and the better the cell activity. The preferred ratio is 3:1.

[0025] Based on the above, this invention uses soy protein isolate or pea protein to replace human serum albumin to avoid ethical controversies, and the degradation cycle is ≤30 days. It uses a biodegradable buffer solution, and the PLGA and sodium alginate composite solution is completely degraded in the natural environment in 28 days to avoid microplastic pollution. At the same time, it uses trehalose and plant polyphenols to replace DMSO to eliminate the risk of chemical toxicity. Furthermore, by reducing cellular energy consumption through metabolic inhibitors (6-aminonicotinamide, 20-50 nM) and inhibiting apoptosis by combining with heat shock proteins, the viability rate was ≥90% after 14 days of storage at room temperature.

[0026] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An environmentally friendly cell preservation method, characterized in that, Includes the following steps: Step 1: Incubate the cells to be preserved in a pretreatment solution containing natural sugar protectants and antioxidants. The natural sugar protectant is trehalose (3-5% w / v), and the antioxidant is plant-extracted polyphenolic compounds (0.1-0.5% w / v). The incubation temperature is 4-37℃, and the time is 30-60 minutes. Step 2: Collect the pretreated cells by centrifugation, wash with phosphate-buffered saline (PBS), and resuspend in environmentally friendly cell preservation solution; Step 3: Store the cell suspension at room temperature (18-25℃).

2. The environmentally friendly cell preservation method according to claim 1, characterized in that: The environmentally friendly cell preservation solution comprises the following components: plant-derived protein substitute (10-30% w / v), low molecular weight heparin calcium (20-50 IU / ml), edaravone (0.1-1% w / v), glutamine (1-3 mM), and biodegradable buffer.

3. The environmentally friendly cell preservation method according to claim 1, characterized in that: The pretreatment solution also includes the metabolic inhibitor 6-aminonicotinamide at a concentration of (20-50 nM).

4. The environmentally friendly cell preservation method according to claim 2, characterized in that: The plant-derived protein substitute is soy protein isolate or pea protein, with a concentration of 15-25% w / v.

5. The environmentally friendly cell preservation method according to claim 2, characterized in that: The biodegradable buffer solution is a composite solution of PLGA and sodium alginate, with a PLGA:sodium alginate ratio of 3:1 and a pH value of 7.0-7.

4.

6. The environmentally friendly cell preservation method according to claim 2, characterized in that: The preservation solution also includes heat shock protein (HSP70, 0.005-0.02% w / v).

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