CN111955454A - Human in-vitro tooth preservative fluid and preparation method thereof - Google Patents
Human in-vitro tooth preservative fluid and preparation method thereof Download PDFInfo
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- CN111955454A CN111955454A CN202010406305.8A CN202010406305A CN111955454A CN 111955454 A CN111955454 A CN 111955454A CN 202010406305 A CN202010406305 A CN 202010406305A CN 111955454 A CN111955454 A CN 111955454A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention discloses a human in vitro tooth preservative fluid and a preparation method thereof, wherein the human in vitro tooth preservative fluid comprises the following components: human serum albumin, astaxanthin, alpha-linolenic acid, sodium citrate, cefepime hydrochloride for injection, amphotericin B, supernatant concentrated solution and sodium chloride injection with the mass concentration of 0.9 percent. The preservation solution has definite components, balanced nutrition and no toxic or side effect on cells, can well maintain the cell activity of the isolated dental pulp at 2-8 ℃, effectively prevents exogenous microbial pollution introduced in the collection and transportation link, effectively prolongs the preservation time, and is beneficial to large-scale production.
Description
Technical Field
The invention relates to a preserving fluid for isolated human teeth.
The invention also relates to a preparation method of the in vitro tooth preservative fluid.
Background
Mesenchymal Stem Cells (MSCs) are a class of primitive cells that have self-renewal capacity and possess multilineage differentiation potential in a suitable microenvironment. It is first found in the bone marrow and then isolated in fat, bone, muscle, lung, liver, pancreas, and in umbilical cord, cord blood, placental tissue, dental pulp, respectively. Because of its strong proliferation ability, strong differentiation ability, low immunogenicity and immunoregulation function, its clinical application value is receiving more and more attention.
At present, most of deciduous teeth and third molar teeth which are taken down by adult orthodontics in the tooth replacement period of children are thrown away as medical wastes, but researches show that dental pulp tissues in dental pulp cavities are rich in a large amount of stem cells, can be self-renewed and multi-directionally differentiated, and have strong replication capacity. Can be used for treating oral diseases such as periodontitis, periodontal regeneration, biological tooth root regeneration, oral cavity bone defect repair, etc., and can repair pathological changes and injuries of tissues and organs, and has the capability of treating various diseases, regulating immunity and resisting aging. And the immunogen is relatively low, the rejection reaction is relatively small, and the vaccine can be used by variant.
However, the dental pulp has little content in the teeth and is easy to shrink, so the preservation solution can effectively maintain the metabolism and cell activity of the dental pulp, prevent exogenous pollution in the collection and transportation links, and lay a foundation for the subsequent separation culture and clinical application of the dental pulp stem cells.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preserving fluid for isolated human teeth.
The invention also provides a preparation method of the in vitro tooth preservative fluid. The preservation solution can effectively maintain the cell activity and the form of dental pulp tissues and maintain the biological characteristics of the dental pulp tissues within 7 days, thereby remarkably improving the separation efficiency of dental pulp stem cells and laying a foundation for clinical treatment of the dental pulp stem cells.
The invention provides the following technical scheme:
a preserving fluid for isolated human teeth comprises the following components: 4-8% of human serum albumin (v/v), 1-10umol/L of astaxanthin (w/v), 4-10ug/ml of alpha-linolenic acid (w/v), 5-10% of sodium citrate (v/v), 0.2-0.5ug/ml of cefepime hydrochloride for injection, 0.01-0.02mg/ml of amphotericin B, supernatant concentrate and sodium chloride injection with the mass concentration of 0.9%.
Preferably, taking the supernatant concentrated solution and sodium chloride injection 1 with mass concentration of 0.9%: 19, diluting and mixing uniformly to obtain the base liquid.
Preferably, the human in vitro tooth preservative fluid comprises the following components: 6% of human serum albumin (v/v), 5umol/L of astaxanthin (w/v), 7ug/ml of alpha-linolenic acid (w/v), 7% of sodium citrate (v/v), 0.4ug/ml of cefepime hydrochloride (w/v) for injection and 0.015mg/ml of amphotericin B.
All reagents used were filtered and sterilized before preparation.
Preparing a supernatant concentrated solution: inoculating P1 dental pulp stem cells in culture flask, adding serum-free culture medium, standing at 37 deg.C under saturated humidity and CO2Culturing in 5% culture box, growing to 80-90% confluence, collecting cell supernatant, performing continuous passage expansion to P5, and collecting supernatant when the cell grows to 80-90% confluence.
Human serum albumin: is the protein with the highest content in plasma, and accounts for 40-60% of the total protein in plasma. Can maintain the plasma colloid osmotic pressure constant and undertake certain transportation function. The albumin is added to provide nutrition for the stem cells and improve the survival rate of the stem cells.
Astaxanthin: as an antioxidant, the antioxidant has a unique molecular structure, has strong capacity of scavenging oxygen free radicals and inhibiting singlet oxygen, and can effectively prevent oxygen atoms from interacting with unsaturated fatty acids of cell membranes and prevent the progress of oxidation reaction, thereby protecting cells and DNA from being damaged by the oxidation reaction. The lipid soluble cell has lipid solubility, can neutralize free radicals in cells, protect proteins in the cells, enable the cells to effectively metabolize, enable the proteins in the cells to better play functions, inhibit the oxidative damage of the free radicals to organisms, can penetrate through the outer walls of the cells, directly remove oxygen radicals in the cells and enhance the regeneration capacity of the cells.
Alpha-linolenic acid: is a basic substance forming cell membranes and biological enzymes, plays a decisive role in human health, and plays a structural function and a regulation function while the alpha linolenic acid is used for energy supply for growth, cell metabolism and muscle movement.
Sodium citrate: also known as sodium citrate, can combine with calcium ions in blood to form chelate, thereby preventing blood coagulation.
Cefepime hydrochloride for injection: the fourth generation of semi-synthetic cephalosporin has stronger antibacterial activity to gram-positive bacteria and gram-negative bacteria and is stable to beta-lactamase.
Amphotericin B: can effectively inhibit and kill fungi.
Supernatant concentrate: the supernatant of the stem cells is a nutrient solution for cell culture, nutrient absorption and factor secretion in the cell proliferation process are exchanged and completed in the supernatant, and cytokines such as platelet-derived growth factor (PDGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), insulin growth factor (IGF-1, IGF-II), Nerve Growth Factor (NGF), interleukin growth factor (IL-1, IL-2, IL-3) and the like secreted by the dental pulp mesenchymal stem cells effectively maintain the vitality of the dental pulp stem cells.
Sodium chloride injection with mass concentration of 0.9%: the commonly used clinical sodium chloride solution with osmotic pressure equal to human plasma. The invention adopts 0.9 percent sodium chloride solution, is in clinical medical grade, is consistent with the osmotic pressure of cells of a human body, and can provide an isotonic environment for the cells as a solvent.
The preparation method of the in vitro tooth preservative fluid for the human comprises the following steps: under the aseptic condition, diluting and uniformly mixing the supernatant concentrated solution and sodium chloride injection with the mass concentration of 0.9% according to the mass ratio of 1:18-20, taking the mixture as a base solution, adding 4-8% of human serum albumin (v/v), 1-10umol/L of astaxanthin (w/v), 4-10ug/ml of alpha-linolenic acid (w/v), 5-10% of sodium citrate (v/v), 0.2-0.5ug/ml of cefepime hydrochloride (w/v) for injection and 0.01-0.02mg/ml of amphotericin B into the base solution, placing the mixture into a sterile bottle, fully mixing the mixture, placing the sterile bottle into a constant temperature shaking table, shaking at 10-15 ℃ and 100-120rpm for 15-20min, preparing the tooth in-vitro preservative solution, and preserving the tooth at 2-8 ℃.
Preferably, taking the supernatant concentrated solution and 0.9% sodium chloride injection according to the mass ratio of 1: 19, diluting and mixing evenly.
Preferably, the human in vitro tooth preservative fluid comprises the following components: 6% of human serum albumin (v/v), 5umol/L of astaxanthin (w/v), 7ug/ml of alpha-linolenic acid (w/v), 7% of sodium citrate (v/v), 0.4ug/ml of cefepime hydrochloride (w/v) for injection and 0.015mg/ml of amphotericin B.
The method for preserving the isolated human teeth comprises the following steps:
the deciduous teeth or the third molar are the third molar which needs to be pulled out in advance by people with proper age of the deciduous teeth and adults due to orthodontic treatment, the teeth are collected in a professional collecting mechanism, the teeth are taken down and disinfected, and then the teeth are placed in a collecting bottle containing preservation liquid to be preserved at the temperature of 2-8 ℃, preferably 4 ℃ for 7 days.
The excised tooth contains healthy deciduous teeth and a third molar. The deciduous teeth or the third molars are third molars which are required to be pulled out in advance by the adult and the suitably aged people of the deciduous teeth and are collected by a professional collecting mechanism.
The in vitro tooth is preserved in the preservation solution of the invention, and can still separate out stem cells after being preserved for 7 days at the temperature of 2-8 ℃, wherein the surface markers of the stem cells are more than 95 percent of CD73, CD90 and CD105, and are less than 2 percent of CD34 and HLA-DR. The cell activity is good.
Compared with the prior art, the invention has the beneficial effects that: the preserving fluid is prepared by taking the supernatant concentrated solution and sodium chloride injection with the mass concentration of 0.9% as base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid, sodium citrate, cefepime hydrochloride for injection and amphotericin B. The preservation solution has definite components, balanced nutrition and no toxic or side effect on cells, can well maintain the cell activity of isolated dental pulp at 2-8 ℃, and maintains the biological characteristics within 7 days, thereby obviously improving the separation efficiency of dental pulp stem cells.
Drawings
FIG. 1: the preservation solution preserved for 7 days, and the stem cells of P0 generation are crawled out from the deciduous tooth pulp tissue block.
FIG. 2: the preservation solution preserved for 7 days, and the P0 generation stem cells crawled out from the ground tooth pulp tissue blocks.
FIG. 3: the storage solution stores the stem cells of deciduous tooth pulp P1 generation for 7 days.
FIG. 4: the storage solution stores 7 days old grinding tooth pulp P1 generation stem cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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
Preparing a supernatant concentrated solution: taking P1 as substitute for dental pulp stem cell at 1 × 105Inoculating to T75 culture flask at density of 16ml, adding serum-free culture medium into each flask, and standing at 37 deg.C under saturated humidity and CO2Culturing in 5% culture box, growing to 80-90% confluence, collecting cell supernatant, performing continuous passage expansion to P5, and collecting supernatant when the cell grows to 80-90% confluence.
And (3) concentrating the supernatant: the collected supernatant was filtered through a 0.22 μm filter, and then the supernatant of the stem cells was concentrated by passing through a Vivaflow 50 swirling/tangential flow ultrafilter having a molecular weight of 50KD, and then the concentrate was passed through a Vivaflow 50 swirling/tangential flow ultrafilter having a molecular weight of 3KD, and the retentate was collected and stored at-20 ℃ or-80 ℃. The concentration ratio of the stem cell supernatant is 38-42 times.
The following examples 2 to 7 are methods for preparing the preservative solutions.
Example 2
Under the aseptic condition, taking the supernatant concentrated solution and sodium chloride injection 1 with the mass concentration of 0.9 percent: 18, diluting and uniformly mixing the mixture to be used as a base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid and sodium citrate into the base solution, and adding cefepime hydrochloride and amphotericin B for injection; the final concentration of the injection is respectively 4 percent of human serum albumin (v/v), 1umol/L of astaxanthin (w/v), 4ug/ml of alpha-linolenic acid (w/v), 5 percent of sodium citrate (v/v), 0.2ug/ml of cefepime hydrochloride for injection and 0.01mg/ml of amphotericin B; placing into a sterile bottle, placing into a constant temperature shaking table, shaking at 10 deg.C and 120rpm for 15min to obtain in vitro tooth preservative solution, and preserving at 2 deg.C.
Example 3
Under the aseptic condition, taking the supernatant concentrated solution and 0.9% of sodium chloride injection 1: 19, diluting and uniformly mixing the mixture to be used as a base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid and sodium citrate into the base solution, and adding cefepime hydrochloride and amphotericin B for injection; the final concentration of the injection is 5 percent of human serum albumin (v/v), 3umol/L of astaxanthin (w/v), 5ug/ml of alpha-linolenic acid (w/v), 6 percent of sodium citrate (v/v), 0.3ug/ml of cefepime hydrochloride for injection and 0.01mg/ml of amphotericin B; placing into a sterile bottle, placing into a constant temperature shaking table, shaking at 10 deg.C and 100rpm for 20min to obtain in vitro tooth preservative solution, and preserving at 4 deg.C.
Example 4
Under the aseptic condition, taking the supernatant concentrated solution and sodium chloride injection 1 with the mass concentration of 0.9 percent: 19, diluting and uniformly mixing the mixture to be used as a base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid and sodium citrate into the base solution, and adding cefepime hydrochloride and amphotericin B for injection; the final concentration of the compound is 6 percent of human serum albumin (v/v), 5umol/L of astaxanthin (w/v), 7ug/ml of alpha-linolenic acid (w/v), 7 percent of sodium citrate (v/v), 0.4ug/ml of cefepime hydrochloride for injection and 0.015mg/ml of amphotericin B (w/v); mixing completely and making into preservative solution. Placing into a sterile bottle, placing into a constant temperature shaking table, shaking at 15 deg.C and 100rpm for 15min to obtain in vitro tooth preservative solution, and preserving at 4 deg.C.
Example 5
Under the aseptic condition, taking the supernatant concentrated solution and sodium chloride injection 1 with the mass concentration of 0.9 percent: 19, diluting and uniformly mixing the mixture to be used as a base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid and sodium citrate into the base solution, and adding cefepime hydrochloride and amphotericin B for injection; the final concentrations of the compounds are 7 percent of human serum albumin (v/v), 8umol/L of astaxanthin (w/v), 8ug/ml of alpha-linolenic acid (w/v), 8 percent of sodium citrate (v/v), 0.4ug/ml of cefepime hydrochloride for injection and 0.015mg/ml of amphotericin B (w/v); placing into a sterile bottle, placing into a constant temperature shaking table, shaking at 15 deg.C and 120rpm for 20min to obtain in vitro tooth preservative solution, and preserving at 6 deg.C.
Example 6
Under the aseptic condition, taking the supernatant concentrated solution and sodium chloride injection 1 with the mass concentration of 0.9 percent: 19, diluting and uniformly mixing the mixture to be used as a base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid and sodium citrate into the base solution, and adding cefepime hydrochloride and amphotericin B for injection; the final concentration of the mixture is respectively 8 percent of human serum albumin (v/v), 9umol/L of astaxanthin (w/v), 9ug/ml of alpha-linolenic acid (w/v), 9 percent of sodium citrate (v/v), 0.5ug/ml of cefepime hydrochloride for injection and 0.02mg/ml of amphotericin B (w/v); placing into a sterile bottle, placing into a constant temperature shaking table, shaking at 12 deg.C and 110rpm for 18min to obtain in vitro tooth preservative solution, and preserving at 7 deg.C.
Example 7
Under the aseptic condition, taking the supernatant concentrated solution and sodium chloride injection 1 with the mass concentration of 0.9 percent: 20, diluting and uniformly mixing the mixture to be used as a base solution, adding human serum albumin, astaxanthin, alpha-linolenic acid and sodium citrate into the base solution, and adding cefepime hydrochloride and amphotericin B for injection; the final concentration of the injection is 8 percent of human serum albumin (v/v), 10umol/L of astaxanthin (w/v), 10ug/ml of alpha-linolenic acid (w/v), 10 percent of sodium citrate (v/v), 0.5ug/ml of cefepime hydrochloride for injection and 0.02mg/ml of amphotericin B (w/v); placing into a sterile bottle, placing into a constant temperature shaking table, shaking at 12 deg.C and 120rpm for 20min to obtain in vitro tooth preservative solution, and preserving at 8 deg.C.
Collecting the isolated human teeth: the donors are negative in HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-treponema pallidum antibody, CMV-IgM, EBV and mycoplasma detection items detected by the three hospitals. The deciduous teeth or the third molar are the third molar which needs to be pulled out in advance by people with proper age of the deciduous teeth and adults due to orthodontic treatment, the teeth are collected in a professional collecting mechanism, the teeth are taken down and disinfected, and then the teeth are placed in a collecting bottle containing preserving fluid to be preserved at the temperature of 2-8 ℃; 10 deciduous teeth and 10 wisdom teeth were respectively extracted and placed in the preservation solution prepared by the method of the present invention in example 4, cells were cultured by tissue block method after being preserved for 7 days in the preservation environment of 2-8 ℃, and the sterility of teeth, the growth of cells and the phenotype of cells were analyzed.
Taking out the teeth from the preservation solution, placing in a sterile dish, washing with 0.9% physiological saline for 2 times, squeezing the teeth with a sterile instrument, and taking out the dental pulp. Placing into a centrifugal tube, and cutting into 1mm with blunt scissors3Inoculating the left and right small blocks into 6-well plate at a density of 0.2g/1 ml/well, culturing, changing the liquid once every 3 days, and observing cellsThe time of the climb-out and the growth of the cells.
As a result, no pollution occurs in 20 stored teeth, compared with fresh teeth (2-3 days of deciduous teeth; 3-4 days of grinding, cells around tissue blocks are observed), the creeping time of deciduous tooth pulp stem cells stored for 7 days is 3-4 days, the creeping time of adult third grinding tooth pulp stem cells is 3-6 days, the creeping cells of deciduous teeth and grinding tooth pulp are in a typical fusiform shape, and the cell nucleus is clearly not obviously different from the fresh tooth pulp stem cells in shape, as shown in figures 1 and 2. The cell reaches over 85% confluency after the tissue block culture for 10-14 days, and the cell ratio is 1: after passage 3, a confluency of more than 85% was reached 2-3 days, as shown in FIGS. C and D. Cells were cultured to P3-5 passages for phenotypic cell testing.
Cell phenotype detection
Centrifuging the P3-P5 generation cell suspension at 1500rpm for 6min, washing the cell precipitate with DPBS for 2 times, and taking 0.8-1 × 106100ul of cell suspension is placed in an EP tube, 15ul of primary antibody (the primary antibody is not added in a blank tube) is added, the cell suspension is incubated for 30min in a dark place at 4 ℃, 400ul of DPBS is added, the cell suspension is centrifuged for 6min at 1500rpm, the supernatant is discarded, 10ul of secondary antibody is added, the cell suspension is incubated for 30min in a dark place at 4 ℃, 400ul of DPBS is added, the cell suspension is centrifuged for 6min at 1500rpm, the supernatant is discarded, 500ul of DPBS is added, the cell suspension is uniformly mixed and then transferred to a flow-type machine-loading tube, and the cell suspension. Results are shown in Table 1.
Table 1: streaming assay results
As can be seen from the detection data in figures 1-4 and table 1, the stem cells extracted from the isolated teeth preserved by the preservation solution are full, have good refractivity and are in typical spindle shapes, and flow detection shows that the cells highly express specific proteins of mesenchymal stem cells such as CD73, CD90, CD105 and the like, the expression rate is more than 95 percent, and the expression rate of CD34 and HLA-DR is less than 2 percent.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A preserving fluid for in vitro teeth of a human is characterized in that: it comprises the following components: 4-8% of human serum albumin (v/v), 1-10umol/L of astaxanthin (w/v), 4-10ug/ml of alpha-linolenic acid (w/v), 5-10% of sodium citrate (v/v), 0.2-0.5ug/ml of cefepime hydrochloride for injection, 0.01-0.02mg/ml of amphotericin B, supernatant concentrate and sodium chloride injection with the mass concentration of 0.9%.
2. The preservation solution for isolated human teeth according to claim 1, wherein: taking the supernatant concentrated solution and sodium chloride injection 1 with the mass concentration of 0.9%: 19, diluting and mixing uniformly to obtain the base liquid.
3. The preservation solution for isolated human teeth according to claim 1, wherein: the human in vitro tooth preservative fluid comprises the following components: 6% of human serum albumin (v/v), 5umol/L of astaxanthin (w/v), 7ug/ml of alpha-linolenic acid (w/v), 7% of sodium citrate (v/v), 0.4ug/ml of cefepime hydrochloride (w/v) for injection and 0.015mg/ml of amphotericin B.
4. The preservation solution for isolated human teeth according to claim 1, wherein: the reagents used were all sterilized by filtration before preparation.
5. A preparation method of a human in vitro tooth preservative fluid is characterized by comprising the following steps: the method comprises the following steps: under the aseptic condition, diluting and uniformly mixing the supernatant concentrated solution and sodium chloride injection with the mass concentration of 0.9% according to the mass ratio of 1:18-20, taking the mixture as a base solution, adding 4-8% of human serum albumin (v/v), 1-10umol/L of astaxanthin (w/v), 4-10ug/ml of alpha-linolenic acid (w/v), 5-10% of sodium citrate (v/v), 0.2-0.5ug/ml of cefepime hydrochloride (w/v) for injection and 0.01-0.02mg/ml of amphotericin B into the base solution, placing the mixture into a sterile bottle, fully mixing the mixture, placing the sterile bottle into a constant temperature shaking table, shaking at 10-15 ℃ and 100-120rpm for 15-20min, preparing the tooth in-vitro preservative solution, and preserving the tooth at 2-8 ℃.
6. The method for preparing a preserving fluid for in vitro human teeth as claimed in claim 5, wherein: taking the supernatant concentrated solution and sodium chloride injection with the mass concentration of 0.9% according to the mass ratio of 1: 19, diluting and mixing evenly.
7. The method for preparing a preserving fluid for in vitro human teeth as claimed in claim 5, wherein: the human in vitro tooth preservative fluid comprises the following components: 6% of human serum albumin (v/v), 5umol/L of astaxanthin (w/v), 7ug/ml of alpha-linolenic acid (w/v), 7% of sodium citrate (v/v), 0.4ug/ml of cefepime hydrochloride (w/v) for injection and 0.015mg/ml of amphotericin B.
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