CN112522190A - High-survival-rate storage and transportation method for umbilical cord tissues - Google Patents
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Abstract
The invention discloses a high-survival-rate storage and transportation method for umbilical cord tissues, which comprises the following operation steps of: (1) taking mesenchymal stem cells, after the culture medium is re-suspended, evenly re-paving the mesenchymal stem cells into four bottles, and harvesting when the cell fusion reaches more than 90%; (2) pouring out the old culture medium in the culture bottle, washing with normal saline, removing the residual culture medium, adding trypsin for digestion, and adding normal saline to stop digestion to obtain cell digestive juice; (3) centrifuging the cell digestive juice, discarding the supernatant, and washing away trypsin to obtain mesenchymal cells; (4) and (4) after the mesenchymal cells are resuspended, repeating the operation in the step (3) to obtain mesenchymal cell sediment, then adding the cell storage and transportation liquid, uniformly mixing, and putting the mixture into a storage and transportation box for cell transportation. The high-survival-rate storage and transportation method for the umbilical cord tissues, provided by the invention, is simple to operate and low in cost, and can effectively ensure the cell survival rate of the umbilical cord mesenchymal stem cells in the storage and transportation process.
Description
Technical Field
The invention belongs to the technical field of cell storage and transportation, and particularly relates to a high-survival-rate storage and transportation method for umbilical cord tissues.
Background
Mesenchymal Stem Cells (MSCs), a pluripotent stem cell present in umbilical cord tissue of a newborn, can differentiate into many kinds of tissue cells. The gene is derived from the mesoderm in the early development stage, and the gene is increasingly concerned by finding that the gene has the characteristics of multidirectional differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation, self-replication and the like. Mesenchymal stem cells are widely present in various tissues of the human body, and mainly comprise: thymus, peripheral blood, dental pulp, amnion, umbilical cord blood, fat, bone marrow, umbilical cord, placenta, etc. The umbilical cord mesenchymal stem cells have the following advantages: (1) the source is rich, the collection is simple and convenient, and no adverse effect is caused to the mother and the newborn; (2) the quantity is large, the multiplication capacity is strong, and the product can be used for many times; (3) the gene is stable, not easy to mutate and safe and reliable to use; (4) the product has low immunogenicity, and has immunoregulatory effect, and can inhibit immune rejection reaction; (5) can be widely used for treating multi-system diseases, beautifying, protecting health and resisting aging
The cells need to be stored in a storage medium for several hours or even longer after leaving the culture environment and being transplanted into a human body, and the selection, storage time and storage temperature of the storage medium have direct influence on the quality and treatment effect of the cells during transplantation. At present, the umbilical cord mesenchymal stem cells are stored and transported in a traditional normal saline mode, namely, the umbilical cord mesenchymal stem cells are digested into single cells and then directly resuspended in normal saline for storage and transportation. The traditional cell storage and transportation method is easy to have the defects of high cell damage and easy cell agglomeration.
Disclosure of Invention
In order to solve the existing problems, the invention provides a high-survival-rate storage and transportation method for umbilical cord tissues.
The invention is realized by the following technical scheme.
A high survival rate storage and transportation method for umbilical cord tissues comprises the following operation steps:
(1) 1mL of the suspension with a density of 6X 105After the culture medium is resuspended, the mesenchymal stem cells per mL are evenly and repeatedly spread into four bottles, each bottle is a 25mL system, and the cells are harvested when the cell fusion reaches more than 90%;
(2) pouring out the old culture medium in the culture bottle, washing with normal saline, removing the residual culture medium, adding 2-4ml trypsin into each bottle for digestion for 25-35s, adding 8-12ml normal saline, and stopping digestion to obtain cell digestive juice;
(3) centrifuging the cell digestive juice, discarding the supernatant, and washing away trypsin to obtain mesenchymal cells;
(4) and (3) after the mesenchymal cells are resuspended, repeating the operation in the step (3) to obtain mesenchymal cell sediment, then adding a cell storage and transportation liquid, uniformly mixing, and putting the mixture into a storage and transportation box for cell transportation, wherein the cell storage and transportation liquid is prepared from the following components in parts by weight: 0.8-1.2 parts of human serum albumin, 85-90 parts of normal saline, 1-3 parts of lentinan, 0.4-0.8 part of astragaloside IV and 400-600 units of heparin sodium.
Specifically, in the step (1), the culture medium contains 8-12% by mass of fetal calf serum, 1.8-2.2mg/L of epidermal growth factor, 1.8-2.2mg/L of fibroblast growth factor and 1.8-2.2mg/L of vascular endothelial growth factor.
Specifically, in the step (2) and the step (4), the mass fraction of sodium chloride in the physiological saline is 0.85-0.90%.
Specifically, in the step (3) and the step (4), the rotation speed during centrifugation is 800-.
Specifically, in the step (4), the solution used for resuspension is PBS buffer.
According to the technical scheme, the beneficial effects of the invention are as follows:
the high-survival-rate storage and transportation method for the umbilical cord tissues, provided by the invention, is simple to operate and low in cost, and can effectively ensure the cell survival rate of the umbilical cord mesenchymal stem cells in the storage and transportation process. The storage and transportation liquid prepared by the invention can be directly used for clinical infusion, meets the requirements of large quantity demand and urgent application of umbilical cord mesenchymal stem cells, can solve the problem of rapid transportation of the mesenchymal stem cells, is the storage and transportation liquid and the injection, can be directly infused into a human body, and achieves rapid transportation and immediate use of the cells on the premise of ensuring the quality of the stem cells. The method for collecting the mesenchymal stem cells can effectively avoid the phenomenon that the mesenchymal stem cells agglomerate in the storage and transportation process.
Drawings
FIG. 1 shows the measurement of cell viability at different times in example 1 and comparative example 1.
FIG. 2 shows the results of flow assays of the phenotype of mesenchymal cells, CD34, CD44, CD45, CD73, CD90, CD105, HLA-DR cells obtained by the method of example 2.
FIG. 3 is a morphological observation result of mesenchymal cells obtained by the method of example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, it will be appreciated by those of ordinary skill in the art that numerous technical details are set forth in order to provide a better understanding of the present invention in its various embodiments. However, the technical solution claimed in the present invention can be implemented without these technical details and various changes and modifications based on the following embodiments.
Example 1
A high survival rate storage and transportation method for umbilical cord tissues comprises the following operation steps:
(1) 1mL of the suspension with a density of 6X 105The mesenchymal stem cells per mL are evenly paved into four bottles after the culture medium is resuspended, each bottle is a 25mL system, and the cells are harvested when the cell fusion reaches more than 90%, wherein the culture medium contains 8% of fetal calf serum by mass and 1.8mg/L of surfaceEpidermal growth factor, fibroblast growth factor of 1.8mg/L, vascular endothelial growth factor of 1.8 mg/L;
(2) pouring out the old culture medium in the culture bottle, washing with normal saline, removing the residual culture medium, adding 2ml of trypsin into each bottle for digestion for 25s, and adding 8ml of normal saline to stop digestion to obtain cell digestive juice;
(3) centrifuging the cell digestive juice, removing supernatant, washing away trypsin to obtain mesenchymal cells, wherein the rotating speed of a centrifuge is 800r/min and the centrifuging time is 8 min;
(4) after being resuspended by PBS buffer solution, the mesenchymal cells are subjected to the operation of the step (3) to obtain mesenchymal cell sediment, then cell storage and transportation liquid is added to be mixed uniformly, and then the mixture is put into a storage and transportation box for cell transportation, wherein the cell storage and transportation liquid is prepared from the following components in parts by weight: 0.8 part of human serum albumin, 85 parts of normal saline, 1 part of lentinan, 0.4 part of astragaloside IV and 400 units of heparin sodium.
In this example, the mass fraction of sodium chloride in the physiological saline was 0.85%.
Example 2
A high survival rate storage and transportation method for umbilical cord tissues comprises the following operation steps:
(1) 1mL of the suspension with a density of 6X 105After the culture medium is resuspended, the mesenchymal stem cells are evenly paved into four bottles, each bottle is a 25mL system, and the mesenchymal stem cells are harvested when the cell fusion reaches more than 90%, wherein the culture medium contains 10% by mass of fetal calf serum, 2.0mg/L of epidermal growth factor, 2.0mg/L of fibroblast growth factor and 2.0mg/L of vascular endothelial growth factor;
(2) pouring out the old culture medium in the culture bottle, washing with normal saline, removing the residual culture medium, adding 3ml of trypsin into each bottle for digestion for 30s, and adding 10ml of normal saline to stop digestion to obtain cell digestive juice;
(3) centrifuging the cell digestive juice, discarding the supernatant, and washing away trypsin to obtain mesenchymal cells, wherein the rotation speed of a centrifuge is 1000r/min and the centrifugation time is 10 min;
(4) after being resuspended by PBS buffer solution, the mesenchymal cells are subjected to the operation of the step (3) to obtain mesenchymal cell sediment, then cell storage and transportation liquid is added to be mixed uniformly, and then the mixture is put into a storage and transportation box for cell transportation, wherein the cell storage and transportation liquid is prepared from the following components in parts by weight: 1.0 part of human serum albumin, 88 parts of normal saline, 2 parts of lentinan, 0.6 part of astragaloside IV and 500 units of heparin sodium.
In this example, the mass fraction of sodium chloride in the physiological saline was 0.88%.
Example 3
A high survival rate storage and transportation method for umbilical cord tissues comprises the following operation steps:
(1) 1mL of the suspension with a density of 6X 105After the culture medium is resuspended, the mesenchymal stem cells are evenly paved into four bottles, each bottle is a 25mL system, and the mesenchymal stem cells are harvested when the cell fusion reaches more than 90%, wherein the culture medium contains 12% by mass of fetal calf serum, 2.2mg/L of epidermal growth factor, 2.2mg/L of fibroblast growth factor and 2.2mg/L of vascular endothelial growth factor;
(2) pouring out the old culture medium in the culture bottle, washing with normal saline, removing the residual culture medium, adding 4ml of trypsin into each bottle for digestion for 35s, and adding 12ml of normal saline to stop digestion to obtain cell digestive juice;
(3) centrifuging the cell digestive juice, removing supernatant, and washing away trypsin to obtain mesenchymal cells, wherein the rotation speed of a centrifuge is 1200r/min and the centrifugation time is 12 min;
(4) after being resuspended by PBS buffer solution, the mesenchymal cells are subjected to the operation of the step (3) to obtain mesenchymal cell sediment, then cell storage and transportation liquid is added to be mixed uniformly, and then the mixture is put into a storage and transportation box for cell transportation, wherein the cell storage and transportation liquid is prepared from the following components in parts by weight: 1.2 parts of human serum albumin, 90 parts of normal saline, 3 parts of lentinan, 0.8 part of astragaloside IV and 600 units of heparin sodium.
In this example, the mass fraction of sodium chloride in the physiological saline was 0.90%.
Comparative example 1
The cell storage and transportation liquid in the step (4) does not contain lentinan, astragaloside IV and heparin sodium, and the other operation steps are completely the same as the embodiment 1.
And (3) testing:
first, test example 1 and comparative example 1 to examine cell viability and apoptosis results at different times
The samples are respectively taken and subjected to trypan blue staining, counting and apoptosis kit flow detection after being placed for 12h, 18h and 24h at 4 ℃ in the labeled group example 1 and the comparative example 1. As shown in fig. 1, the trypan blue staining count results showed that the cell survival rate of example 1 was higher than that of comparative example 1 at each time period after 12h and 18 h.
Flow cytometry detection of umbilical cord mesenchymal stem cells
By adopting the method of example 2, sampling is carried out after standing for 24h, the culture medium is re-suspended and inoculated into a T75 culture flask after centrifugation, and after the cell fusion degree reaches 90%, the cell is harvested, and the cell is subjected to flow detection of cell phenotypes such as CD34, CD44, CD45, CD73, CD90, CD105 and HLA-DR, as shown in FIG. 2, all samples well meet the criteria of mesenchymal stem cells (CD34+, CD45+, HLA-DR is lower than 2%; CD44+, CD73+, CD90+, CD105+ is higher than 95%).
Third, example 3 Observation of cell morphology after reattachment of umbilical cord mesenchymal stem cells stored and transported
Using the method of example 3, samples were taken after 24h of standing and passaged at 1X 104/cm2The cell density of the umbilical cord MSC can be generally increased to 90% confluency after about 72 hours of inoculation, as shown in figure 3, the cell density becomes a single triangle or fusiform after passage, dense cell clone gradually appears in a bottle, the umbilical cord MSC grows in a uniform fusiform adherent manner and is closely arranged in a parallel or vortex shape.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should understand that they can make various changes, modifications, additions and substitutions within the spirit and scope of the present invention.
Claims (5)
1. A high survival rate storage and transportation method of umbilical cord tissues is characterized by comprising the following operation steps:
(1) 1mL of the suspension with a density of 6X 105After the culture medium is resuspended, the mesenchymal stem cells per mL are evenly and repeatedly spread into four bottles, each bottle is a 25mL system, and the cells are harvested when the cell fusion reaches more than 90%;
(2) pouring out the old culture medium in the culture bottle, washing with normal saline, removing the residual culture medium, adding 2-4ml trypsin into each bottle for digestion for 25-35s, adding 8-12ml normal saline, and stopping digestion to obtain cell digestive juice;
(3) centrifuging the cell digestive juice, discarding the supernatant, and washing away trypsin to obtain mesenchymal cells;
(4) and (3) after the mesenchymal cells are resuspended, repeating the operation in the step (3) to obtain mesenchymal cell sediment, then adding a cell storage and transportation liquid, uniformly mixing, and putting the mixture into a storage and transportation box for cell transportation, wherein the cell storage and transportation liquid is prepared from the following components in parts by weight: 0.8-1.2 parts of human serum albumin, 85-90 parts of normal saline, 1-3 parts of lentinan, 0.4-0.8 part of astragaloside IV and 400-600 units of heparin sodium.
2. The high survival rate storage and transportation method for umbilical cord tissue as claimed in claim 1, wherein in the step (1), the culture medium contains 8-12% by mass of fetal bovine serum, 1.8-2.2mg/L of epidermal growth factor, 1.8-2.2mg/L of fibroblast growth factor, and 1.8-2.2mg/L of vascular endothelial growth factor.
3. The high survival rate storage and transportation method for umbilical cord tissue as claimed in claim 1, wherein in the step (2) and the step (4), the mass fraction of sodium chloride in the physiological saline is 0.85-0.90%.
4. The method as claimed in claim 1, wherein the rotation speed during centrifugation in steps (3) and (4) is 800-1200r/min, and the centrifugation time is 8-12 min.
5. The method for storing and transporting umbilical cord tissue with high survival rate as claimed in claim 1, wherein the resuspension solution used in step (4) is PBS buffer.
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