CN109497038A - A kind of fat saves liquid and preparation method thereof - Google Patents

Abstract

The invention discloses a fat preservation solution, which is used for preserving fat tissue samples. Atrol, Icariin, Hydrocortisone, VEGF, IGF‑I, EPO. The fat preservation solution of the invention is easy to prepare, low in cost and convenient to use, can maintain the activity of stem cells in adipose tissue for a long time, greatly reduces the time limit from collection to preparation of tissue samples, and can effectively reduce the probability of contamination.

Description

A kind of fat preservation liquid and preparation method thereof

technical field

The invention relates to the field of biotechnology, in particular to a preservation solution for preserving fat samples.

Background technique

Mesenchymal stem cells (MSCs) are a kind of stem cells with multi-directional differentiation potential, which have the potential to differentiate into various mesodermal cell lineages, including osteoblasts, adipocytes, chondrocytes, etc. In the normal process of injury repair in the body, MSCs can be recruited to the injury site under the induction of chemokines, proliferate and differentiate locally, and participate in injury repair and tissue regeneration through paracrine action. Adipose tissue-derived MSCs are easy to isolate and expand in vitro, and maintain multi-directional differentiation potential after multiple divisions and passages. They are ideal seed cells for cell therapy and regenerative medicine. Adipose tissue is widely distributed, easy to collect, has no ethical issues, is basically painless, and can be collected multiple times.

The preparation process of adipose MSCs includes many steps such as tissue sample collection, transportation, handover, detection, separation, cryopreservation, recovery, primary culture and subculture. Three steps of transportation, handover and detection are required after the collection of fat samples and before separation. , it needs to be stored for a long time in actual operation. Once the adipose tissue sample is collected, it is separated from the original in vivo environment, and the activity of MSCs in it will be affected by many factors, such as time, temperature, osmotic pressure and so on. At present, adipose tissue samples need to be collected from different places in many cases, and the three steps of transportation, handover and detection take longer, and the tissue samples need to be stored for a long time, and the influencing factors are more complicated. In the domestic biological field, the preservation of adipose tissue samples mostly adopts the method of direct bottling or supplementation of basal medium. The storage time of directly bottled adipose tissue samples is generally less than 24 hours, which brings considerable time pressure to the transportation, handover and detection of adipose tissue samples, and there is an urgent need to improve the storage method.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the problems of short storage time of fat samples and affected stem cell acquisition rate and activity in the prior art, and to provide a fat preservation solution with simple ratio, low cost and convenient use. The adipose preservation solution of the present invention can preserve the activity of stem cells in the adipose tissue sample, and can be effectively preserved for at least 120 hours under the condition of 0-8° C. The activity of the isolated stem cells is little affected by time, which greatly reduces the time from the collection of the tissue sample. The preparation time is limited, and the probability of contamination can be effectively reduced.

In order to achieve the above purpose, the technical scheme adopted in the present invention is: a fat preservation solution, each liter of fat preservation solution is composed of the following components by weight: 13.4g of high-sugar DMEM dry powder medium, 2.438g of sodium bicarbonate, 2-10mg of insulin , Penicillin 100～200mg, Streptomycin 100～200mg, Amphotericin 2.5～5mg, Aspirin 200～400mg, Vitamin C 300～700mg, Resveratrol 7～12mg, Icariin 2～10mg, Hydrocortisone 20～40ug, VEGF 10～20μg, IGF-I 5～10μg, EPO 5～10μg, the balance is water for injection.

Above-mentioned a kind of fat preservation liquid, its preparation method comprises the following steps:

(1) Pour 13.4g of high-sugar DMEM dry powder medium into a container, wash off the residual medium in the bag with a small amount of water for injection, and incorporate it into the container. Add water for injection to 950ml, stir gently to dissolve;

(2) add 2.438 g sodium bicarbonate;

(3) Slightly stir to dissolve, add water for injection to 1L;

(4) adjust pH value to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution;

(5) Insulin 2-10 mg, penicillin 100-200 mg, streptomycin 100-200 mg, amphotericin 2.5-5 mg, aspirin 200-400 mg, vitamin C 300-700 mg, resveratrol 7-12 mg, Carriin 2～10mg, Hydrocortisone 20～40ug, VEGF 10～20μg, IGF-I 5～10μg, EPO 5～10μg, mix well;

(6) Filtration and sterilization through a 0.22um filter membrane to obtain the fat preservation solution of the present invention, which is then packaged and frozen for storage; it can be thawed at 0-8° C. in advance before use.

The fat preservation solution of the present invention, wherein high sugar DMEM and sodium bicarbonate maintain the osmotic balance of cells inside and outside the tissue, maintain the pH value, maintain the moist state of the adipose tissue and provide nutrients; penicillin, streptomycin, and amphotericin can prevent bacteria, Mold and other pollution, and can eliminate the pollution that has occurred; vitamin C, resveratrol, icariin are antioxidants, which can maintain fat activity; hydrocortisone inhibits immunity and protects stem cell activity in adipose tissue samples; insulin promotes Absorption and utilization of sugar by fat; cytokines such as VEGF, IGF-I, EPO, can maintain and increase the activity of stem cells in adipose tissue.

The fat preservation solution of the invention is simple to prepare, low in cost and convenient to use, so that the collected tissue samples can effectively maintain the activity of stem cells in the tissue samples for a long time during transportation, handover, detection before separation and the like, and can prevent and remove the tissue samples. pollution and improve efficiency.

Detailed ways

The present invention will be described in further detail below.

Example 1

The fat preservation solution provided by the present invention is composed of the following components by weight per liter of fat preservation solution: 13.4 g of high-sugar DMEM dry powder medium, 2.438 g of sodium bicarbonate, 10 mg of insulin, 200 mg of penicillin, 200 mg of streptomycin, 200 mg of amphotericin Vitamin C 5mg, Aspirin 400mg, Vitamin C 700mg, Resveratrol 12mg, Icariin 10mg, Hydrocortisone 40ug, VEGF 20μg, IGF-I 10μg, EPO 10μg, the balance is water for injection.

The preparation method of the fat preservation solution is as follows:

(1) Pour 13.4g of high-sugar DMEM dry powder medium (brand: GIBCO, product number 12100-046) into a container, wash the residual medium in the bag with a small amount of water for injection, and incorporate it into the container. Add water for injection to 950ml, stir gently to dissolve;

(2) add 2.438 g sodium bicarbonate;

(3) Slightly stir to dissolve, add water for injection to 1L;

(4) adjust pH value to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution;

(5) Insulin 10mg (brand Sigma, product number I0908), penicillin 200mg (brand Amresco, product number 0242), streptomycin 200mg (brand Amresco, product number 0382), amphotericin 5mg (brand Amresco, product number E437), Aspirin 400mg (Sigma, A2093-100G), Vitamin C 700mg (Sigma, A5960-10mg), Resveratrol 12mg (Sigma, R5010-100MG), Icariin 10mg (Shanghai Microcrystalline Bio, 489-32-7 , 20mg), hydrocortisone 40ug (Sigma, H3160), VEGF 20μg (PeproTech, 96-100-20-2), IGF-I 10μg (PeproTech, 96-100-11-20), EPO 10μg (PeproTech, CYT-201), mix well;

(6) Filter and sterilize with a 0.22um filter membrane to obtain a fat preservation solution, which is then packaged and frozen for storage; it can be thawed in advance at 0~8°C before use.

Embodiment 2

A fat preservation solution provided by the present invention is composed of the following components by weight per liter of fat preservation solution: 13.4 g of high-sugar DMEM dry powder medium, 2.438 g of sodium bicarbonate, 2 mg of insulin, 100 mg of penicillin, 100 mg of streptomycin, 100 mg of amphotericin Vitamin C 2.5mg, aspirin 200mg, vitamin C 300mg, resveratrol 7mg, icariin 2mg, hydrocortisone 20ug, VEGF 10μg, IGF-I 5μg, EPO 5μg, the balance is water for injection.

The preparation method of the fat preservation solution is as follows:

(1) Pour 13.4g of high-sugar DMEM dry powder medium into a container, wash the residual medium in the bag with a small amount of water for injection, and put it into the container. Add water for injection to 950ml, stir gently to dissolve;

(2) add 2.438 g sodium bicarbonate;

(3) Slightly stir to dissolve, add water for injection to 1L;

(4) adjust pH value to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution;

(5) Insulin 2mg, penicillin 100mg, streptomycin 100mg, amphotericin 2.5mg, aspirin 200mg, vitamin C 300mg, resveratrol 7mg, icariin 2mg, hydrocortisone 20ug, VEGF 10μg were added in sequence , IGF-I 5μg, EPO 5μg, mix well;

(6) Filter and sterilize with a 0.22um filter membrane, sub-pack, freeze and store to obtain a fat preservation solution; thaw at 0~8°C in advance before use.

Embodiment 3

A fat preservation solution provided by the present invention, each liter of fat preservation solution is composed of the following components by weight: high-sugar DMEM dry powder medium 13.4g, sodium bicarbonate 2.438g, insulin 5mg, penicillin 150mg, streptomycin 150mg, amphoteric mold Vitamin C 4mg, Aspirin 300mg, Vitamin C 400mg, Resveratrol 8mg, Icariin 3mg, Hydrocortisone 30ug, VEGF 15μg, IGF-I 6μg, EPO 6μg, the balance is water for injection.

The preparation method of the fat preservation solution is as follows:

(1) Pour 13.4g of high-sugar DMEM dry powder medium into a container, wash off the residual medium in the bag with a small amount of water for injection, and incorporate it into the container. Add water for injection to 950ml, stir gently to dissolve;

(2) add 2.438 g sodium bicarbonate;

(3) Slightly stir to dissolve, add water for injection to 1L;

(4) adjust pH value to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution;

(5) Insulin 5mg, penicillin 150mg, streptomycin 150mg, amphotericin 4mg, aspirin 300mg, vitamin C 400mg, resveratrol 8mg, icariin 3mg, hydrocortisone 30ug, VEGF 15μg, IGF-I 6μg, EPO 6μg, mix well;

(6) Filtration and sterilization of 0.22um filter membrane to obtain fat preservation solution, subpackage, and cryopreservation; it can be thawed at 0~8℃ in advance before use.

Embodiment 4

Collection and preservation of adipose tissue: use the fat preservation solution of the present invention to preserve the human adipose tissue after liposuction and before separation, that is, after aseptically collecting fat, aspirating and discarding the swelling fluid, and placing the collected adipose tissue into a adipose tissue containing the present invention. In the fat collection bottle of the fat preservation solution, tighten the bottle cap and seal it, and put it in an incubator at 0~8°C for constant temperature preservation until separation.

Comparison of the preservation effect of the preservation solution: the collected adipose tissue is divided into 5 parts, and they are respectively stored in the fat preservation solution of the present invention, phosphate buffered saline PBS, physiological saline and high-glucose DMEM medium (liquid). The adipose tissue was directly placed alone, stored at a constant temperature of 0~8°C for 24 hours, 48 hours, 72 hours, and 120 hours, and then digested and separated by collagenase. The nucleated cells obtained by taking the same grams of adipose tissue blocks were routinely cultured for 9 days, and then digested. count. At the same time, a portion of fresh adipose tissue was not preserved as a control, and the nucleated cells obtained from the same gram of adipose tissue block after direct collagenase digestion and separation were routinely cultured for 9 days, and the count after digestion was 6.20×10 ⁷ .

The save result is displayed as follows:

Contamination rate statistics for fat collection and transportation in the past year, 2 out of 64 adipose tissues stored without any solution were contaminated, with a contamination rate of 3.1%; none of the 371 adipose tissues preserved by the fat preservation solution of the present invention pollution, the pollution rate is 0.

It can be seen that the fat preservation solution of the present invention can effectively maintain the activity of stem cells in the collected tissue samples for a long time (more than 5 days) in the process of transportation, handover, detection before separation, etc. Yield, and can prevent and remove contamination, expanding the business scope of tissue sample bank stem cell storage.

Claims (2)

1. a kind of fat saves liquid, characterized in that every liter of fat saves liquid and is made of by weight following ingredient: DMEM in high glucose dry powder Culture medium 13.4g, sodium bicarbonate 2.438g, 2~10mg of insulin, 100~200mg of penicillin, 100~200mg of streptomysin, two 2.5~5mg of property mycin, 200 ~ 400mg of aspirin, 300~700mg of vitamin C, 7~12mg of resveratrol, icariin 2 ~10mg, 20~40ug of hydrocortisone, 10~20 μ g of VEGF, 5 IGF-I~10 μ g, 5 EPO~10 μ g, surplus are injection Use water.

2. a kind of fat according to claim 1 saves liquid, preparation method includes the following steps:

(1) DMEM in high glucose dehydrated medium 13.4g is poured into a container, with a small amount of water for injection by remaining medium in bag It washes down, is incorporated to container；Inject water to 950ml, gentle agitation dissolution；

(2) 2.438 g sodium bicarbonates are added；

(3) gentle agitation dissolves, and injects water to 1L；

(4) pH value is adjusted to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution；

(5) 2~10mg of insulin, 100~200mg of penicillin, 100~200mg of streptomysin, anphotericin are successively separately added into 2.5~5mg, 200 ~ 400mg of aspirin, 300~700mg of vitamin C, 7~12mg of resveratrol, icariin 2~ 10mg, 20~40ug of hydrocortisone, 10~20 μ g of VEGF, 5 IGF-I~10 μ g, 5 EPO~10 μ g, mix well；

(6) degerming of 0.22um membrane filtration is made present invention fat and saves liquid, packing, freezen protective；It is placed in 0 when use in advance ~ 8 DEG C of defrostings.