CN111548988A - Medical rinsing liquid and preparation method and application thereof - Google Patents

Medical rinsing liquid and preparation method and application thereof Download PDF

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CN111548988A
CN111548988A CN202010405909.0A CN202010405909A CN111548988A CN 111548988 A CN111548988 A CN 111548988A CN 202010405909 A CN202010405909 A CN 202010405909A CN 111548988 A CN111548988 A CN 111548988A
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rinsing liquid
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vitamin
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CN111548988B (en
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王学庆
王楠
王嘉轩
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Jiangsu Zhihai Biotechnology Co ltd
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Abstract

The invention provides a medical rinsing liquid and a preparation method and application thereof, belonging to the technical field of medical reagents. The medical rinsing liquid comprises glucose, vitamins, basic fibroblast growth factors, keratinocyte growth factors, hepatocyte growth factors, vascular endothelial growth factors, leptin and buffer; the concentrations of the glucose, the vitamins, the basic fibroblast growth factor, the keratinocyte growth factor, the hepatocyte growth factor, the vascular endothelial growth factor and the leptin are 0.5-4 g/L, 0.002-0.02 g/L, 0.005-0.05 mg/L, 0.002-0.02 mg/L, 0.02-0.25 mg/L, 0.04-0.4 mg/L and 0.002-0.02 mg/L in sequence; the pH value of the medical rinsing liquid is 7.2-7.5. The medical rinsing liquid provided by the invention can effectively ensure the activity of cells or tissues.

Description

Medical rinsing liquid and preparation method and application thereof
Technical Field
The invention relates to the technical field of medical reagents, in particular to a medical rinsing liquid and a preparation method and application thereof.
Background
In the in vitro treatment process of cells or tissues, rinsing liquid is generally needed to wash the cells or tissues so as to remove impurities on the basis of ensuring the activity of the cells or tissues and enable subsequent related treatment to be smoothly carried out. Therefore, the rinsing liquid has an important influence on the smooth proceeding of the in vitro treatment process of the cells or tissues.
At present, the fat particle injection autograft technology is widely applied to soft tissue filling, and has the advantages of good biocompatibility, convenient acquisition, abundant sources, simple operation, good filling effect, low cost and the like. However, the absorption rate of the fat particles after the injection and the autografting of the fat particles is high, the volume and the weight of the transplanted fat particles are reduced by more than 50 percent, the effect after the fat transplantation is difficult to predict, and the survival rate of the fat transplantation is low because the non-survival cells are easy to liquefy, necrose and infect.
The survival rate of fat transplantation is related to many factors, and the steps of obtaining adipose tissues, treating in vitro, injecting and the like all have certain influence on the survival rate of fat transplantation. Fat particles in the fat particle injection autograft technology are usually obtained by negative pressure liposuction under the condition of swelling anesthesia, but at present, no consensus is provided for how to treat fat cells, and when part of plastic doctors wash the fat cells, normal saline (clinically, ringer's solution can also be used to replace the normal saline) is adopted, so that the survival rate is still to be improved.
Disclosure of Invention
The medical rinsing liquid provided by the invention adopts the combined action of a plurality of growth factors and is matched with an energy substance and a buffer system, so that the activity of cells or tissues can be effectively ensured, and the survival rate of autologous fat transplantation can be effectively improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a medical rinsing liquid, which comprises glucose, vitamins, basic fibroblast growth factors, keratinocyte growth factors, hepatocyte growth factors, vascular endothelial growth factors, leptin and a buffer solution;
wherein the concentration of glucose is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L;
the solvent of the buffer solution is water;
the pH value of the medical rinsing liquid is 7.2-7.5.
Preferably, the concentration of glucose in the medical rinsing liquid is 0.5-2 g/L, the concentration of vitamin is 0.002-0.01 g/L, the concentration of basic fibroblast growth factor is 0.005-0.03 mg/L, the concentration of keratinocyte growth factor is 0.005-0.015 mg/L, the concentration of hepatocyte growth factor is 0.04-0.15 mg/L, the concentration of vascular endothelial growth factor is 0.08-0.2 mg/L, and the concentration of leptin is 0.005-0.015 mg/L.
Preferably, the concentration of glucose in the medical rinsing liquid is 1g/L, the concentration of vitamin is 0.002g/L, the concentration of basic fibroblast growth factor is 0.01mg/L, the concentration of keratinocyte growth factor is 0.01mg/L, the concentration of hepatocyte growth factor is 0.05mg/L, the concentration of vascular endothelial growth factor is 0.1mg/L, and the concentration of leptin is 0.01 mg/L.
Preferably, the vitamins comprise vitamin B, vitamin C, vitamin K1 and vitamin E, and the mass ratio of the vitamin B to the vitamin C to the vitamin K1 to the vitamin E is 1:1:1: 1.
Preferably, the buffer solution is phosphate buffer solution, and the buffer solution comprises NaCl, KCl and NaHCO3、Na2HPO4、KH2PO4And water; the pH value of the phosphate buffer solution is 7.2-7.5.
The invention provides a preparation method of the medical rinsing liquid, which comprises the following steps:
mixing the components of the medical rinsing liquid and sterilizing to obtain the rinsing liquid.
The invention provides application of the medical rinsing liquid in the scheme or the medical rinsing liquid prepared by the preparation method in the scheme in culture, transfer, storage or rinsing of cells or tissues.
Preferably, the cells comprise stem cells or adipocytes and the tissue comprises adipose tissue.
Preferably, the method of rinsing comprises the steps of:
and mixing the medical rinsing liquid with the cells or tissues to be treated, rinsing, and then removing the liquid in the obtained system to complete the rinsing of the cells or tissues.
Preferably, the rinsing is performed for 2-3 times, and the volume ratio of the medical rinsing liquid to the cells or tissues to be treated is 3-4: 10 in the single rinsing process.
The invention provides a medical rinsing liquid, which comprises glucose, vitamins, basic fibroblast growth factors, keratinocyte growth factors, hepatocyte growth factors, vascular endothelial growth factors, leptin and a buffer solution; wherein the concentration of glucose is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L; the solvent of the buffer solution is water; the pH value of the medical rinsing liquid is 7.2-7.5. The medical rinsing liquid provided by the invention adopts the combined action of various growth factors and is matched with an energy substance and a buffer system, so that the activity of cells or tissues can be effectively ensured, and the survival rate of autologous fat transplantation can be effectively improved. The results of the examples show that when the fat particles are rinsed by the rinsing liquid provided by the invention, the cell proliferation rate is obviously higher than that of the fat particles rinsed by normal saline; compared with normal saline containing 1% of double antibody, the rinsing liquid provided by the invention is used for rinsing adipose tissues and then culturing, so that primary cells can adhere to the wall and stretch more quickly.
Drawings
FIG. 1 is a graph showing the comparison of cell proliferation rates of adipocytes obtained by using the experimental group and the control group of example 1, cultured under the same conditions;
FIG. 2 is a graph showing a comparison of cell growth states after adipose stem cells obtained from the experimental group and the control group of application example 2 were isolated and cultured under the same conditions for 3 days.
Detailed Description
The invention provides a medical rinsing liquid, which comprises glucose, vitamins, basic fibroblast growth factors, keratinocyte growth factors, hepatocyte growth factors, vascular endothelial growth factors, leptin and a buffer solution; the solvent of the buffer solution is water; the pH value of the medical rinsing liquid is 7.2-7.5. In the invention, the concentration of glucose in the medical rinsing liquid is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L; preferably, the concentration of glucose in the medical rinsing liquid is 0.5-2 g/L, the concentration of vitamin is 0.002-0.01 g/L, the concentration of basic fibroblast growth factor is 0.005-0.03 mg/L, the concentration of keratinocyte growth factor is 0.005-0.015 mg/L, the concentration of hepatocyte growth factor is 0.04-0.15 mg/L, the concentration of vascular endothelial growth factor is 0.08-0.2 mg/L, and the concentration of leptin is 0.005-0.015 mg/L; more preferably, the concentration of glucose in the medical rinsing liquid is 1g/L, the concentration of vitamin is 0.002g/L, the concentration of basic fibroblast growth factor is 0.01mg/L, the concentration of keratinocyte growth factor is 0.01mg/L, the concentration of hepatocyte growth factor is 0.05mg/L, the concentration of vascular endothelial growth factor is 0.1mg/L, and the concentration of leptin is 0.01 mg/L.
In the present invention, the basic fibroblast growth factor, the keratinocyte growth factor, the hepatocyte growth factor, the vascular endothelial growth factor and the leptin can be natural growth factors (or leptin) and can also be recombinant growth factors (or leptin); in the examples of the present invention, recombinant growth factors (or leptin) are specifically used.
In the present invention, the vitamins preferably include vitamin B, vitamin C, vitamin K1 and vitamin E, and the mass ratio of vitamin B, vitamin C, vitamin K1 and vitamin E is preferably 1:1:1: 1. In the present invention, the vitamins can function as co-enzyme factors (e.g., vitamin K and most vitamin B) and biological antioxidants (e.g., vitamin C and vitamin E), which are beneficial for maintaining cell viability and stability.
In the present invention, the buffer is preferably phosphate buffer, the pH value of the phosphate buffer is preferably 7.4, and the phosphate buffer preferably comprises NaCl, KCl and NaHCO in composition3、Na2HPO4、KH2PO4And water; the pH value of the phosphate buffer solution is preferably 7.2-7.5; in the invention, more preferably, the concentration of NaCl in the medical rinsing liquid is 8g/L, the concentration of KCl is 0.4g/L, and NaHCO is used3Has a concentration of 0.35g/L, Na2HPO4The concentration of (A) is 0.1g/L, KH2PO4The concentration of (3) was 0.06 g/L. In the present invention, the buffer solution can provide a necessary buffer environment for extraction and purification of cells, tissues, or the like. In the present invention, it is preferable to use a phosphate buffer solution which can remove blood, anesthetic solution, fiber fragments, etc. mixed in cells or tissues when the cells or tissues are rinsed; meanwhile, the stability of the cells of the fat cells and the mesenchymal stem cells in vitro can be improved, the integrity and the activity of the fat cells can be maintained, and the survival rate of cell transplantation can be improved.
In the invention, the basic fibroblast growth factor (bFGF) can promote the hyperplasia of preadipocytes and connective tissues, promote the revascularization process and is beneficial to the survival of adipocytes; keratinocyte Growth Factor (KGF) and Vascular Endothelial Growth Factor (VEGF) can promote mitosis of endothelial cells, promote regeneration of nerve vessels around transplanted tissues and improve survival rate of fat cells; the Hepatocyte Growth Factor (HGF) has strong functions of promoting angiogenesis and establishing collateral circulation, and is beneficial to improving the survival rate of autologous fat transplantation; leptin is mainly secreted by fat cells, can promote the transformation of preadipocytes to mature fat cells, vascular endothelial cells have Leptin receptors, and the Leptin can promote the proliferation of the vascular endothelial cells, accelerate local vascular proliferation, increase local blood supply and enhance the vascular proliferation of transplanted tissues.
In the present invention, the glucose is used as an energy substance and can provide a necessary nutrient environment for extraction and purification of adipocytes, tissues, and the like.
The invention adopts the combined action of various growth factors and is matched with an energy substance and a buffer system, so that the survival rate of autologous fat transplantation can be effectively improved.
In the present invention, the water is preferably purified water.
The invention provides a preparation method of the medical rinsing liquid, which comprises the following steps: mixing the components of the medical rinsing liquid and sterilizing to obtain the rinsing liquid.
In the invention, after the components of the medical rinsing liquid are mixed according to the proportion, the pH value of the obtained mixed liquid is 7.2-7.5, and HCl or NaOH can be further adopted to adjust the pH value of the mixed liquid according to the requirement. The present invention is not particularly limited, and the sterilization may be performed by a method known to those skilled in the art; the invention preferably adopts filtration sterilization, and particularly, the components of the medical rinsing liquid are mixed and then pass through a 0.22 mu m filter membrane to realize sterilization. After sterilization is completed, the present invention is preferably packaged under helium protection (to avoid oxidation of unstable components) to obtain a rinse solution.
The invention provides the application of the medical rinsing liquid in the technical scheme or the medical rinsing liquid prepared by the preparation method in the technical scheme in the culture, transfer, storage or rinsing of cells or tissues. The type of the cells or tissues is not particularly limited, and any cells or tissues that need to be treated in vitro (cultured, transferred, stored or rinsed) may be used, and specifically, the cells may be adipocytes or stem cells, and the stem cells may be adipose stem cells or other stem cells, which is not particularly limited in the present invention; the tissue may be adipose tissue. The medical rinsing liquid provided by the invention can be applied to fat transplantation operations of dermatology, plastic surgery and medical cosmetology, and can be specifically used for in vitro treatment of extracted fat cells and in vitro treatment of stem cells in the process of stem cell transplantation.
In the present invention, the rinsing method preferably includes the steps of: and mixing the medical rinsing liquid with the cells or tissues to be treated, rinsing, and then removing the liquid in the obtained system to complete the rinsing of the cells or tissues. In the invention, the rinsing frequency is preferably 2-3 times, and in the single rinsing process, the volume ratio of the medical rinsing liquid to the cells or tissues to be treated is preferably 3-4: 10. In the embodiment of the invention, specifically, cells or tissues to be treated are loaded into an injector, the injector is used for absorbing the medical rinsing liquid, the medical rinsing liquid is turned over for 3-5 times up and down, and then the medical rinsing liquid is kept stand for 5-15 min to remove the lower layer liquid, so that 1 rinsing is completed; and (4) repeatedly rinsing for 1-2 times according to the operation to obtain the purified cells or tissues. The method provided by the invention is adopted to rinse the cells or tissues, the loss rate of the cells or tissues is less than 5% after the whole rinsing process is finished, and the cell survival rate is high.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In this example, the recombinant bFGF, KGF, HGF, VEGF and Leptin used were purchased from R & D Systems, and the trade names and the trade numbers were as follows:
recombinant bFGF: the trade name is Recombinant Human FGF basic, and the commercial product number is 233-FB-025;
recombinant KGF: the product is called Recombinant Human KGF/FGF-7Protein, and the product number is 251-KG-050;
recombinant HGF: the trade name is Recombinant Human HGF Protein, and the trade name is 294-HG-025;
recombinant VEGF: the trade name is Recombinant Human VEGF, and the commercial product number is 293-VE-050
And (3) recombinant Leptin: the trade name is Recombinant Human leptin protein, and the trade name is 398-LP-01M.
Weighing the following raw materials in concentration: NaCl 8g/L, KCl 0.4g/L, NaHCO30.35g/L, Na2HPO40.1g/L, KH2PO4Is 0.06g/L and glucose is 1gL, 0.5mg/L of vitamin B, 0.5mg/L of vitamin C, 0.5mg/L of vitamin E, 0.5mg/L of vitamin K1, 0.01mg/L of bFGF, 0.01mg/L of KGF, 0.05mg/L of HGF, 0.1mg/L of VEGF, 0.01mg/L of Leptin and the balance of purified water;
the preparation method comprises the following steps: mixing the above materials, adjusting pH to 7.4, filtering for sterilization (with 0.22 μm filter membrane), introducing helium gas for protection, and packaging to obtain rinsing solution.
Application example 1
Rinsing of adipocytes
Experimental groups: adopting a syringe to suck under negative pressure to obtain fat particles, filling 50mL of the fat particles into the syringe, sucking 15mL of rinsing liquid prepared in the embodiment 1 by using the syringe, turning over the rinsing liquid for 4 times up and down, standing for 10min to remove lower-layer liquid, and finishing rinsing for 1 time; the rinsing was repeated 2 more times according to the above procedure to obtain purified fat particles.
Control group: the above procedure was followed except that the rinsing solution prepared in example 1 was replaced with physiological saline.
Taking the purified fat particles of the experimental group and the control group, and diluting the fat particles with DMEM and 10% FBS culture medium until the cell number is 5 × 104And each well is inoculated into a 96-well plate according to 100 mu L per well, after 24 hours of pre-culture, CCK-8 detection is carried out, specifically, 10 mu L of CCK-8 solution is added into each well, incubation is carried out for 2 hours, the absorbance at 450nm is measured, the cell proliferation rate is calculated, and the result is shown in figure 1, wherein 'normal saline' in figure 1 corresponds to a control group, and 'rinsing liquid' corresponds to an experimental group. As is clear from FIG. 1, after the adipocytes were treated with the rinse solution prepared in example 1 and cultured under the same conditions, the cell growth rate was 135% (P) of that of the control group<0.01)。
Application example 2
Isolated culture of adipose-derived stem cells
Experimental groups: the adipose tissue mass was sufficiently sucked by the rinsing liquid prepared in example 1, connective tissues and small blood vessels on the adipose tissue mass were removed by ophthalmic scissors, and then the adipose tissue mass was rinsed 3 times by the rinsing liquid according to the method of application example 1, and the rinsed adipose tissue mass was placed on a sterile gauze, and the adipose tissue mass was dried (concretely, the adipose tissue mass was dried)Drying the adipose tissue blocks in an aseptic environment until no obvious rinsing liquid trace exists on the surface); adding pure calf serum to the dried adipose tissue pieces, and rapidly cutting the adipose tissue pieces to about 1mm3Small tissue blocks of (2) are separately planted at 25cm2In a culture flask, the cells were cultured in an inverted state in a carbon dioxide incubator at 37 ℃.
Control group: the procedure was followed except that the rinsing solution prepared in example 1 was replaced with a physiological saline containing 1% of double antibody.
The cells cultured for 3 days in the experimental group and the control group were observed by a microscope, and the results are shown in FIG. 2, in which "physiological saline" corresponds to the control group and "rinsing solution" corresponds to the experimental group. As can be seen from FIG. 2, the primary cells attached to the adipose tissues and expanded more rapidly than the control group when cultured after rinsing the adipose tissues with the rinse solution provided by the present invention. The continuous culture shows that the primary cells rinsed by the physiological saline containing 1% of double antibody are paved on the bottom of the bottle for 15 days, and the primary cells rinsed by the rinsing liquid provided by the invention can be paved on the bottom of the bottle for 10 days. This shows that the rinsing liquid provided by the invention can promote the growth of the adipose-derived stem cells and maintain the activity of the adipose-derived stem cells.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A medical rinsing liquid is characterized by comprising glucose, vitamins, basic fibroblast growth factors, keratinocyte growth factors, hepatocyte growth factors, vascular endothelial growth factors, leptin and buffer solution;
wherein the concentration of glucose is 0.5-4 g/L, the concentration of vitamin is 0.002-0.02 g/L, the concentration of basic fibroblast growth factor is 0.005-0.05 mg/L, the concentration of keratinocyte growth factor is 0.002-0.02 mg/L, the concentration of hepatocyte growth factor is 0.02-0.25 mg/L, the concentration of vascular endothelial growth factor is 0.04-0.4 mg/L, and the concentration of leptin is 0.002-0.02 mg/L;
the solvent of the buffer solution is water;
the pH value of the medical rinsing liquid is 7.2-7.5.
2. The medical rinsing liquid as claimed in claim 1, wherein the concentration of glucose in the medical rinsing liquid is 0.5-2 g/L, the concentration of vitamin is 0.002-0.01 g/L, the concentration of basic fibroblast growth factor is 0.005-0.03 mg/L, the concentration of keratinocyte growth factor is 0.005-0.015 mg/L, the concentration of hepatocyte growth factor is 0.04-0.15 mg/L, the concentration of vascular endothelial growth factor is 0.08-0.2 mg/L, and the concentration of leptin is 0.005-0.015 mg/L.
3. The medical rinsing liquid as claimed in claim 2, wherein the concentration of glucose in the medical rinsing liquid is 1g/L, the concentration of vitamin is 0.002g/L, the concentration of basic fibroblast growth factor is 0.01mg/L, the concentration of keratinocyte growth factor is 0.01mg/L, the concentration of hepatocyte growth factor is 0.05mg/L, the concentration of vascular endothelial growth factor is 0.1mg/L, and the concentration of leptin is 0.01 mg/L.
4. The medical rinsing liquid as claimed in any one of claims 1 to 3, wherein the vitamins include vitamin B, vitamin C, vitamin K1 and vitamin E, and the mass ratio of the vitamin B to the vitamin C to the vitamin K1 to the vitamin E is 1:1:1: 1.
5. The medical rinse solution of claim 1, wherein the buffer is a phosphate buffer comprising NaCl, KCl, NaHCO3、Na2HPO4、KH2PO4And water; the pH value of the phosphate buffer solution is 7.2-7.5.
6. A method for preparing a medical rinsing liquid as claimed in any one of claims 1 to 5, which comprises the steps of:
mixing the components of the medical rinsing liquid and sterilizing to obtain the rinsing liquid.
7. Use of the medical rinsing fluid according to any one of claims 1 to 5 or the medical rinsing fluid prepared by the preparation method according to claim 6 in culturing, transferring, storing or rinsing cells or tissues.
8. The use of claim 7, wherein the cells comprise stem cells or adipocytes and the tissue comprises adipose tissue.
9. Use according to claim 7 or 8, characterized in that the method of rinsing comprises the following steps:
and mixing the medical rinsing liquid with the cells or tissues to be treated, rinsing, and then removing the liquid in the obtained system to complete the rinsing of the cells or tissues.
10. The application of claim 9, wherein the rinsing is performed 2-3 times, and the volume ratio of the medical rinsing liquid to the cells or tissues to be treated in a single rinsing process is 3-4: 10.
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US20090004160A1 (en) * 2006-01-27 2009-01-01 ByungSoon Park Mass Producing Method of Growth Factor Using Adipose Derived Adult Stem Cells
US20120315254A1 (en) * 2010-02-23 2012-12-13 Sebana Medical Ltd. Methods and compositions for enhancing fat graft survival
CN103977395A (en) * 2013-02-07 2014-08-13 玛旺干细胞医学生物科技股份有限公司 Growth factor preparation and its production method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090004160A1 (en) * 2006-01-27 2009-01-01 ByungSoon Park Mass Producing Method of Growth Factor Using Adipose Derived Adult Stem Cells
US20120315254A1 (en) * 2010-02-23 2012-12-13 Sebana Medical Ltd. Methods and compositions for enhancing fat graft survival
CN103977395A (en) * 2013-02-07 2014-08-13 玛旺干细胞医学生物科技股份有限公司 Growth factor preparation and its production method

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