CN115067318B - Dental pulp mesenchymal stem cell preservation solution and application thereof - Google Patents
Dental pulp mesenchymal stem cell preservation solution and application thereof Download PDFInfo
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- CN115067318B CN115067318B CN202210163429.7A CN202210163429A CN115067318B CN 115067318 B CN115067318 B CN 115067318B CN 202210163429 A CN202210163429 A CN 202210163429A CN 115067318 B CN115067318 B CN 115067318B
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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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W90/00—Enabling technologies or technologies with a potential or indirect contribution to greenhouse gas [GHG] emissions mitigation
- Y02W90/10—Bio-packaging, e.g. packing containers made from renewable resources or bio-plastics
Abstract
The invention discloses dental pulp mesenchymal stem cell preservation solution which comprises the following raw materials in parts by weight: 90-100 parts of 0.9% sodium chloride solution, 1-5 parts of penicillin, 3-6 parts of sodium hyaluronate, 5-10 parts of dipotassium hydrogen phosphate, 1.5-3 parts of 2-hydroxybenzylamine, 1-3 parts of ethyl nicotinate, 1-5 parts of polyinosinic acid and vitamin K 4 5.5-8.5 parts. The invention provides 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K in dental pulp mesenchymal stem cell preservation solution 4 The components of the extract can effectively maintain the activity of the dental pulp mesenchymal stem cells under the preservation condition of 0-6 ℃, and has important significance for the short-term preservation of the dental pulp mesenchymal stem cells in vitro. The preservation solution disclosed by the invention is clear and safe in components, effectively reduces the cost of in-vitro preservation of dental pulp mesenchymal stem cells, and provides convenience for short-term preservation and transportation of dental pulp mesenchymal stem cells. The invention also provides application of the preservation solution, which is used for preservation of dental pulp mesenchymal stem cells, and the preservation process is simple and convenient and easy to operate, thereby providing convenience for scientific research and clinical application of the cells.
Description
Technical Field
The invention relates to the field of stem cells, in particular to dental pulp mesenchymal stem cell preservation solution and application thereof.
Background
Mesenchymal stem cells (mesenchymal stem cell MSC) are multipotent stem cells that can form a variety of cell types. MSCs were first found in bone marrow and subsequently found in a wide variety of tissues during human development and development. With the increasing maturation of mesenchymal stem cells and their related technologies, clinical studies have been conducted in many fields.
At present, mesenchymal stem cells have been isolated from tissues such as bone marrow, umbilical cord, cord blood, placenta, fat, etc., and most used are bone marrow-derived mesenchymal stem cells. However, bone marrow-derived mesenchymal stem cells have problems such as difficult cell collection, serious injury to human body, and age limitation. The mesenchymal stem cells separated from umbilical cord blood, umbilical cord and placenta tissues not only keep the biological characteristics of the mesenchymal stem cells, but also have stronger proliferation and differentiation capacity, and have no harm to the body during collection, but have great limitation on the collection time of tissue materials.
The dental pulp mesenchymal stem cells have all biological characteristics of the mesenchymal stem cells, and are convenient to obtain materials, such as the dental pulp mesenchymal stem cells are abundant in teeth which are naturally shed by children aged 6-11 and wisdom teeth which are needed to be extracted by adults. The dental pulp mesenchymal stem cells have low immunogenicity, can be used without strict pairing, can not cause strong rejection reaction, have an immunoregulatory function, and can not cause rejection reaction after allograft transplantation.
In the prior art, a mode of directly preserving teeth is adopted for preserving dental pulp mesenchymal stem cells, but because the number of dental pulp mesenchymal stem cells separated from teeth is small, a long-time expansion culture is needed to obtain enough cells, and the time needed for the process is long. The expanded cells are directly frozen in liquid nitrogen at low temperature, and the recovery of the cells is a long-term preservation method when the method is applied, but the freezing and preservation of the dental pulp mesenchymal stem cells to be used in a short period by adopting liquid nitrogen are complicated, so that the preservation cost is increased. The existing low-temperature preservation solution generally has the problems that the cell activity cannot be guaranteed, the preservation condition is strictly required, and the like, and is not beneficial to reducing the short-term preservation cost of dental pulp mesenchymal stem cells. Therefore, it is necessary to develop a dental pulp mesenchymal stem cell preservation solution, which can realize the short-term preservation of dental pulp mesenchymal stem cells in vitro and reduce the preservation cost of cells under the condition of ensuring the cell activity.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide an dental pulp mesenchymal stem cell preservation solution, so that the dental pulp mesenchymal stem cells can be preserved in vitro for a short period, and the preservation cost is reduced under the condition of ensuring the cell activity.
The second object of the present invention is to provide an application of the preservation solution.
One of the purposes of the invention is realized by adopting the following technical scheme:
the dental pulp mesenchymal stem cell preservation solution consists of the following raw materials in parts by weight: 90-100 parts of 0.9% sodium chloride solution, 1-5 parts of penicillin, 3-6 parts of sodium hyaluronate, 5-10 parts of dipotassium hydrogen phosphate, 1.5-3 parts of 2-hydroxybenzylamine, 1-3 parts of ethyl nicotinate, 1-5 parts of polyinosinic acid and vitamin K 4 5.5-8.5 parts。
Further, the dental pulp mesenchymal stem cell preservation solution consists of the following raw materials in parts by weight: 95 parts of sodium chloride solution with mass concentration of 0.9%, 2.5 parts of penicillin, 5 parts of sodium hyaluronate, 8 parts of dipotassium hydrogen phosphate, 2 parts of 2-hydroxybenzylamine, 2 parts of ethyl nicotinate, 3.5 parts of polyinosinic acid and vitamin K 4 7 parts.
The second purpose of the invention is realized by adopting the following technical scheme:
the preservation solution is applied to preservation of dental pulp mesenchymal stem cells.
Further, the preservation temperature is 0-6 ℃.
Further, the density of cells in the preservation solution was 5X 10 6 -1×10 7 And each mL.
Further, the application of the dental pulp mesenchymal stem cell preservation solution comprises the following steps:
(1) Penicillin, sodium hyaluronate, dipotassium hydrogen phosphate, 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K are added into 0.9% sodium chloride solution 4 Mixing uniformly
(2) And (3) placing the dental pulp mesenchymal stem cell preservation solution prepared in the step (1) into an environment of 0-6 ℃ for pre-cooling, then adding the dental pulp mesenchymal stem cells to be preserved into the preservation solution, and continuing to preserve at the condition of 0-6 ℃.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an dental pulp mesenchymal stem cell preservation solution, wherein the components such as 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and the like in the preservation solution effectively maintain the activity of dental pulp mesenchymal stem cells under the preservation condition of 0-6 ℃, and the dental pulp mesenchymal stem cell preservation solution has important significance for the short-term preservation of dental pulp mesenchymal stem cells in vitro. The preservation solution disclosed by the invention is clear and safe in components, effectively reduces the cost of in-vitro preservation of dental pulp mesenchymal stem cells, and provides convenience for short-term preservation and transportation of dental pulp mesenchymal stem cells.
The invention also provides application of the preservation solution, which is used for preservation of dental pulp mesenchymal stem cells, and the preservation process is simple and convenient and easy to operate, thereby providing convenience for scientific research and clinical application of the cells.
Drawings
FIG. 1 is a graph showing proliferation curves of dental pulp mesenchymal stem cells after 48 hours of preservation and dental pulp mesenchymal stem cells without preservation in example 1 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.
Sources of dental pulp mesenchymal stem cells are as follows: collecting wisdom teeth of adult aged 25-30 years old, cleaning with PBS, clamping to split teeth, taking dental pulp tissue out with sterile forceps, adding type I collagenase for digestion, adding complete culture medium to stop digestion, centrifuging, adding DMEM/F12 culture medium containing 10% FBS, and re-suspending to cell density of 1×10 4 individual/mL, inoculated into 12-well plates at 37℃in 5% CO 2 After the cells grow to 80% confluence, they are digested with 0.25% pancreatin, and subcultured, and the subcultured P3, P4, P5 generation dental pulp mesenchymal stem cells are taken for preservation.
Example 1
The dental pulp mesenchymal stem cell preservation solution consists of the following raw materials in parts by weight: 95 parts of sodium chloride solution with mass concentration of 0.9%, 2.5 parts of penicillin, 5 parts of sodium hyaluronate, 8 parts of dipotassium hydrogen phosphate, 2 parts of 2-hydroxybenzylamine, 2 parts of ethyl nicotinate, 3.5 parts of polyinosinic acid and vitamin K 4 7 parts.
The application of the preservation solution comprises the following steps:
(1) Penicillin, sodium hyaluronate, dipotassium hydrogen phosphate, 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K are added into 0.9% sodium chloride solution 4 Mixing uniformly
(2) Pre-cooling the dental pulp mesenchymal stem cell preservation solution prepared in the step (1) in an environment of 6 ℃, and then adding the P3 generation dental pulp mesenchymal stem cells into the preservation solution, wherein the density of the cells in the preservation solution is 5 multiplied by 10 6 And (3) keeping the sample at 6 ℃ per mL.
Example 2
The dental pulp mesenchymal stem cell preservation solution consists of the following raw materials in parts by weight: 90 parts of 0.9% sodium chloride solution, 1 part of penicillin, 3 parts of sodium hyaluronate, 5 parts of dipotassium hydrogen phosphate, 1.5 parts of 2-hydroxybenzylamine, 1 part of ethyl nicotinate, 1 part of polyinosinic acid and vitamin K 4 5.5 parts.
The application of the preservation solution comprises the following steps:
(1) Penicillin, sodium hyaluronate, dipotassium hydrogen phosphate, 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K are added into 0.9% sodium chloride solution 4 Mixing uniformly
(2) Pre-cooling the dental pulp mesenchymal stem cell preservation solution prepared in the step (1) in an environment of 4 ℃, and then adding the P4-generation dental pulp mesenchymal stem cells into the preservation solution, wherein the density of the cells in the preservation solution is 8 multiplied by 10 6 8/mL, and the storage at 4 ℃ is continued.
Example 3
The dental pulp mesenchymal stem cell preservation solution consists of the following raw materials in parts by weight: 100 parts of 0.9% sodium chloride solution, 5 parts of penicillin, 6 parts of sodium hyaluronate, 10 parts of dipotassium hydrogen phosphate, 3 parts of 2-hydroxybenzylamine, 3 parts of ethyl nicotinate, 5 parts of polyinosinic acid and vitamin K 4 8.5 parts.
The application of the preservation solution comprises the following steps:
(1) Penicillin, sodium hyaluronate, dipotassium hydrogen phosphate, 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K are added into 0.9% sodium chloride solution 4 Mixing uniformly
(2) Pre-cooling the dental pulp mesenchymal stem cell preservation solution prepared in the step (1) in an environment of 0 ℃, and then adding the P5 generation dental pulp mesenchymal stem cells into the preservation solution, wherein the density of the cells in the preservation solution is 1 multiplied by 10 7 And (3) keeping the sample at 0 ℃ per mL.
Comparative example 1
Comparative example 1 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: the 2-hydroxybenzylamine was omitted and the remainder was the same as in example 1.
Comparative example 2
Comparative example 2 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: ethyl nicotinate was omitted and the remainder was the same as in example 1.
Comparative example 3
Comparative example 3 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: the ethyl nicotinate was replaced with nicotinic acid, and the rest was the same as in example 1.
Comparative example 4
Comparative example 4 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: the ethyl nicotinate was replaced by nicotinamide, and the remainder was the same as in example 1.
Comparative example 5
Comparative example 5 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: polyinosinic acid was omitted, and the rest was the same as in example 1.
Comparative example 6
Comparative example 6 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: inosinic acid was replaced with inosine, and the other was the same as in example 1.
Comparative example 7
Comparative example 7 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: vitamin K 4 Replacement with vitamin K 3 The remainder were the same as in example 1.
Comparative example 8
Comparative example 8 provides a dental pulp mesenchymal stem cell preservation solution, which is different from example 1 in that: vitamin K 4 Replacement with vitamin K 1 The remainder were the same as in example 1.
For examples 1 to 3, the dental pulp mesenchymal stem cells stored in comparative examples 1 to 8 were sampled at 0h, 24h, 48h, respectively, and cell viability was counted using trypan blue staining, and the results are shown in table 1.
TABLE 1
| Sample of | Cell viability at 0h | 24h cell viability | Cell viability for 48h |
| Example 1 | 98.03％ | 94.79％ | 85.61％ |
| Example 2 | 98.54％ | 93.37％ | 83.15％ |
| Example 3 | 97.27％ | 93.06％ | 84.38％ |
| Comparative example 1 | 97.83％ | 84.72％ | 72.47％ |
| Comparative example 2 | 98.02％ | 82.15％ | 71.96％ |
| Comparative example 3 | 98.19％ | 79.32％ | 65.49％ |
| Comparative example 4 | 97.33％ | 85.79％ | 72.07％ |
| Comparative example 5 | 98.04％ | 80.26％ | 61.48％ |
| Comparative example 6 | 97.63％ | 86.27％ | 73.99％ |
| Comparative example 7 | 96.97％ | 89.57％ | 79.38％ |
| Comparative example 8 | 97.66％ | 85.84％ | 71.25％ |
It can be seen from table 1 that the pulp mesenchymal stem cells in examples 1 to 3 had a cell viability of 93% or more at 24 hours of storage, and a cell viability of 83% or more after 48 hours of storage, which was higher than that of comparative examples 1 to 8, and the compositions of the preservation solutions were adjusted in comparative examples 1 to 8, respectively.
The 2-hydroxybenzylamine was omitted in comparative example 1, and the viability of the cells was decreased to a different extent with the prolongation of the preservation time, and the viability of the cells after 48 hours of preservation was 72.47%, which was lower than 85.61% in the examples, indicating that the addition of 2-hydroxybenzylamine helped to maintain the activity of the cells in the preservation solution.
The omission of ethyl nicotinate in comparative example 2, the replacement of ethyl nicotinate with niacin and niacinamide in comparative example 3 and comparative example 4, respectively, wherein the survival rate of cells after the replacement of ethyl nicotinate with niacin in comparative example 3 is less than that of comparative example 2, indicates that niacin has no significant effect on maintaining the activity of dental pulp mesenchymal stem cells. The cell viability was higher than that of comparative example 2, but lower than that of comparative example 1 after the replacement of niacinamide with niacinamide in comparative example 4, indicating that the effect of niacinamide addition was less than that of niacinamide.
In comparative example 5, the inosinic acid was omitted, and in comparative example 6, the replacement of inosinic acid with inosine decreased the survival rate of the cells to a different extent, and in comparative example 6, the survival rate was higher than in comparative example 5, but unlike example 1, it was demonstrated that inosine was less effective in maintaining the activity of dental pulp mesenchymal stem cells than inosinic acid.
Vitamin K was added to comparative example 7 and comparative example 8, respectively 4 Replacement with vitamin K 3 And vitamin K 1 The viability of the cells was also somewhat reduced compared to example 1, in which vitamin K was added 1 The survival rate of the preservation solution cells is the lowest, which indicates vitamin K 4 Has better effect in maintaining the activity of dental pulp mesenchymal stem cells.
In summary, 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K in the preservation solution of the invention 4 The components of the extract can effectively maintain the activity of the dental pulp mesenchymal stem cells under the preservation condition of 0-6 ℃, and has important significance for the short-term preservation of the dental pulp mesenchymal stem cells in vitro. The preservation solution disclosed by the invention is clear and safe in components, effectively reduces the cost of in-vitro preservation of dental pulp mesenchymal stem cells, and provides convenience for short-term preservation and transportation of dental pulp mesenchymal stem cells.
The dental pulp mesenchymal stem cells preserved for 48 hours in example 1 were inoculated into a DMEM/F12 medium containing 10% FBS, and the cell density was 1X 10 4 Individual/mL, at 37 ℃,5% CO 2 Is cultured in an incubator for 7 daysThe number of dental pulp mesenchymal stem cells was counted every day using the non-preserved P3-generation dental pulp mesenchymal stem cells as a control group, and a cell growth curve was drawn, and the result is shown in fig. 1.
As can be seen from FIG. 1, compared with the control group, the cells preserved for 48 hours provided by the invention are equivalent to the control group in proliferation rate, and the number of the cells is slightly reduced compared with the control group, which indicates that the preservation solution provided by the invention has no influence on proliferation activity of dental pulp mesenchymal stem cells, and the preserved cells can still be used for normal proliferation culture.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (6)
1. The dental pulp mesenchymal stem cell preservation solution is characterized by comprising the following raw materials in parts by weight: 90-100 parts of 0.9% sodium chloride solution, 1-5 parts of penicillin, 3-6 parts of sodium hyaluronate, 5-10 parts of dipotassium hydrogen phosphate, 1.5-3 parts of 2-hydroxybenzylamine, 1-3 parts of ethyl nicotinate, 1-5 parts of polyinosinic acid and vitamin K 4 5.5-8.5 parts.
2. The dental pulp mesenchymal stem cell preservation solution according to claim 1, which is characterized by comprising the following raw materials in parts by weight: 95 parts of sodium chloride solution with mass concentration of 0.9%, 2.5 parts of penicillin, 5 parts of sodium hyaluronate, 8 parts of dipotassium hydrogen phosphate, 2 parts of 2-hydroxybenzylamine, 2 parts of ethyl nicotinate, 3.5 parts of polyinosinic acid and vitamin K 4 7 parts.
3. Use of the preservation solution according to claim 1 for preservation of dental pulp mesenchymal stem cells.
4. The use of the dental pulp mesenchymal stem cell preservation solution according to claim 3, wherein the preservation temperature is 0-6 ℃.
5. According toThe use of a preservation solution for dental pulp mesenchymal stem cells according to claim 3, wherein the density of cells in the preservation solution is 5×10 6 -1×10 7 And each mL.
6. Use of a dental pulp mesenchymal stem cell preservation solution according to claim 3, comprising the steps of:
(1) Penicillin, sodium hyaluronate, dipotassium hydrogen phosphate, 2-hydroxybenzylamine, ethyl nicotinate, polyinosinic acid and vitamin K are added into 0.9% sodium chloride solution 4 Mixing uniformly
(2) And (3) placing the dental pulp mesenchymal stem cell preservation solution prepared in the step (1) into an environment of 0-6 ℃ for pre-cooling, then adding the dental pulp mesenchymal stem cells to be preserved into the preservation solution, and continuing to preserve at the condition of 0-6 ℃.
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