CN112056307A - Umbilical cord preservation liquid and umbilical cord preservation method - Google Patents
Umbilical cord preservation liquid and umbilical cord preservation method Download PDFInfo
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- CN112056307A CN112056307A CN202010858237.9A CN202010858237A CN112056307A CN 112056307 A CN112056307 A CN 112056307A CN 202010858237 A CN202010858237 A CN 202010858237A CN 112056307 A CN112056307 A CN 112056307A
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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
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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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Abstract
The invention discloses an umbilical cord preservation solution and an umbilical cord preservation method, wherein the umbilical cord preservation solution comprises the following components in terms of 1L of preservation solution: 15-20 mmol of hyperbranched polyglycerol, 99-101 mmol of lactobionic acid, 24-26 mmol of monopotassium phosphate, 4-6 mmol of magnesium sulfate, 4-6 mmol of adenosine, 2-4 mmol of glutathione reduced form, 6-10 mg of dexamethasone, 0.5-1.5 mmol of allopurinol, 50-70 mmol of sucrose, 40-60 g of dextran-4040, 18-22 mg of isobodidine, 4-6 ml of sodium hydroxide and 19-21 ml of potassium hydroxide. When the umbilical cord is stored, the cleaned umbilical cord is cut into small sections with the length of 3-5 cm, then the small sections are immersed into the storage liquid, and the umbilical cord is stored in the environment with the temperature of 3-5 ℃. The preservation solution can effectively solve the problem that the umbilical cord can not be preserved for a long time after being isolated.
Description
Technical Field
The invention belongs to the technical field of umbilical cord preservation, and particularly relates to an umbilical cord preservation solution and an umbilical cord preservation method.
Background
Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, are derived from early-developing mesoderm, belong to pluripotent stem cells, and are originally found in bone marrow, and are stromal fibroblast stem cells, which are increasingly paid attention to due to their characteristics of multipotentiality, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. Previous studies have shown that MSCs have low immunogenicity, are able to evade immunodetection, and have immunosuppressive properties and tissue repair/regeneration effects.
Bone marrow-derived MSCs are the most deeply studied mesenchymal stem cells, but since the procedures for isolating bone marrow MSCs are complicated and the availability of the mesenchymal stem cells may cause trauma to the donor, the number, proliferation and differentiation capacity of the obtained MSC cells are significantly reduced as the donor ages, and these factors limit the applications of bone marrow-derived MSCs. Therefore, in recent years, it has become an important direction for stem cell research to obtain MSCs from waste fetal tissues (such as placenta, umbilical cord blood, etc.). Related researches also prove that the MSC from the umbilical cord and the MSC from the marrow have the same immunological characteristics and have similar application prospects. Compared with bone marrow-derived MSC, the umbilical cord-derived MSC has the advantages of being easier to separate, culture and proliferate. The umbilical cord MSC has more abundant sources, has higher proliferation capacity and in vitro passage capacity in vitro culture, has no ethical problems, and particularly can meet the requirement of large amount of MSC required by clinical treatment by establishing an umbilical cord MSC library, so that the umbilical cord MSC library has more advantages in clinical application.
However, the isolation of MSCs is currently performed shortly after the umbilical cord leaves the mother, otherwise sufficient primary MSCs cannot be isolated, resulting in the influence of subsequent MSC culture and proliferation. Most of the existing umbilical cords are soaked and stored in 4 ℃ physiological saline or lactated ringer's solution, the storage time is short, and the umbilical cords need to be treated in a short time. The isolation, culture and proliferation of MSC all need high-grade sterility and a laboratory with related conditions to operate, so the requirement on the transportation time of the umbilical cord is higher. In order to prolong the preservation and transportation time of the umbilical cord and ensure the separation, culture and proliferation effects of MSC, the search of a new preservation solution is very important.
Disclosure of Invention
Aiming at the prior art, the invention provides an umbilical cord preservation solution and an umbilical cord preservation method, which aim to solve the problem that an umbilical cord cannot be preserved for a long time after being separated from a body.
In order to achieve the purpose, the invention adopts the technical scheme that: provided is an umbilical cord preservation solution, wherein the preservation solution of the present invention comprises, by volume of 1L:
15-20 mmol of hyperbranched polyglycerol, 99-101 mmol of lactobionic acid, 24-26 mmol of monopotassium phosphate, 4-6 mmol of magnesium sulfate, 4-6 mmol of adenosine, 2-4 mmol of glutathione reduced form, 6-10 mg of dexamethasone, 0.5-1.5 mmol of allopurinol, 50-70 mmol of sucrose, 40-60 g of dextran-4040, 18-22 mg of isobodidine, 4-6 ml of sodium hydroxide and 19-21 ml of potassium hydroxide.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the preservation solution of the present invention includes, in a volume of 1L:
18mmol of hyperbranched polyglycerol, 100mmol of lactobionic acid, 25mmol of monopotassium phosphate, 5mmol of magnesium sulfate, 5mmol of adenosine, 3mmol of glutathione reduced form, 8mg of dexamethasone, 1mmol of allopurinol, 60mmol of sucrose, 4050 g of dextran, 20mg of isobinder, 5ml of sodium hydroxide and 20ml of potassium hydroxide.
Further, the pH of the preservation solution was 7.45. + -. 0.10, and the osmotic pressure was 310. + -. 10 mOSM/L.
When the umbilical cord is preserved by adopting the preservation solution, the preservation process comprises the following steps:
s1: taking a newborn umbilical cord, sequentially cleaning the newborn umbilical cord with alcohol and a PBS solution, and dividing the cleaned umbilical cord into small sections of 3-5 cm;
s2: putting the segmented umbilical cord into a storage container, adding the umbilical cord storage solution according to any one of claims 1 to 3 until the umbilical cord is completely submerged, and then placing the storage container in an environment at 3-5 ℃ for storage.
The invention has the beneficial effects that:
compared with the common preservation solution, the preservation solution of the invention can still separate MSC from the umbilical cord after 7 days of preservation, and the capability of osteogenic differentiation, fibroblast differentiation and chondrogenic differentiation of the MSC separated from the umbilical cord after 7 days of preservation is similar to that of the MSC separated from the fresh umbilical cord. Flow cytometry analysis shows that the cells obtained by umbilical cord separation after 1-day and 7-day storage highly express CD73+ and CD90+, but do not express CD 34-and CD45-, and accord with the immunophenotype of MSC.
Drawings
FIG. 1 is a microscopic tissue morphology 6-8 days after the seeding;
FIG. 2 is a microscopic tissue morphology after 10-12 days from the seeding;
FIG. 3 shows the result of the differentiation study of the fiber forming of the umbilical cord mesenchymal stem cell generation P1;
FIG. 4 shows the result of examining chondrogenic differentiation of umbilical cord mesenchymal stem cells generation P1;
FIG. 5 shows the results of osteogenic differentiation studies of umbilical cord mesenchymal stem cells generation P1;
FIG. 6 shows the expression of CD90+ in cells;
FIG. 7 shows the expression of CD73+ in cells;
FIG. 8 shows the expression of CD 45-in cells;
FIG. 9 shows the expression of CD 34-in cells.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1
An umbilical cord preservation solution comprising, in terms of 1L of preservation solution:
18mmol of hyperbranched polyglycerol, 100mmol of lactobionic acid, 25mmol of monopotassium phosphate, 5mmol of magnesium sulfate, 5mmol of adenosine, 3mmol of glutathione reduced form, 8mg of dexamethasone, 1mmol of allopurinol, 60mmol of sucrose, 4050 g of dextran, 20mg of isobinder, 5ml of sodium hydroxide (50mmol/L) and 20ml of potassium hydroxide (50 mmol/L).
The pH of the preservation solution in this example was about 7.45 and the osmotic pressure was about 310 mOSM/L.
Example 2
An umbilical cord preservation solution comprising, in terms of 1L of preservation solution:
15mmol of hyperbranched polyglycerol, 101mmol of lactobionic acid, 24mmol of monopotassium phosphate, 6mmol of magnesium sulfate, 4mmol of adenosine, 4mmol of glutathione reduced form, 6mg of dexamethasone, 1.5mmol of allopurinol, 50mmol of sucrose, 4060 g of dextran, 18mg of isoboldine, 6ml of sodium hydroxide (50mmol/L) and 19ml of potassium hydroxide (50 mmol/L).
The pH of the preservation solution in this example was about 7.35 and the osmotic pressure was about 300 mOSM/L.
Example 3
An umbilical cord preservation solution comprising, in terms of 1L of preservation solution:
20mmol of hyperbranched polyglycerol, 99mmol of lactobionic acid, 26mmol of monopotassium phosphate, 4mmol of magnesium sulfate, 6mmol of adenosine, 2mmol of glutathione reduced form, 10mg of dexamethasone, 0.5mmol of allopurinol, 70mmol of sucrose, 4040 g of dextran, 22mg of isobledine, 4ml of sodium hydroxide (50mmol/L) and 21ml of potassium hydroxide (50 mmol/L).
The pH of the preservation solution in this example was about 7.55 and the osmotic pressure was about 320 mOSM/L.
Examples of the experiments
The umbilical cords were preserved with the preservation solutions of examples 1 to 3, respectively, and after 7 days, the MSCs were removed and isolated. Since the preservation effects of the preservation solutions of examples 1 to 3 are almost the same, the preservation effect of the preservation solution of the present invention will be described by taking the preservation solution of example 1 as an example.
(1) Umbilical cord preservation
The method comprises the following steps when the umbilical cord is preserved:
s1: taking a newborn umbilical cord, sequentially cleaning the newborn umbilical cord with alcohol and a PBS solution, and dividing the cleaned umbilical cord into small sections of 3-5 cm;
s2: putting the segmented umbilical cord into a storage container, adding the umbilical cord storage solution according to any one of claims 1 to 3 until the umbilical cord is completely submerged, and then placing the storage container in an environment at about 4 ℃ for storage.
(2) Umbilical cord stem cell separation and culture after 7-day storage
The method for separating and culturing the umbilical cord stem cells comprises the following steps:
s1: wiping the clean bench with 75% medical alcohol, and sequentially placing the sterilized experimental devices into the clean bench;
s2: taking out the umbilical cord preserved in the umbilical cord preservation solution, cutting out the umbilical cord with the length of about 1cm, putting the umbilical cord into a culture disc, adding about 10mL of PBS buffer solution, splitting the umbilical cord with sterile scissors, finding out one umbilical cord vein and two umbilical arteries, carefully removing blood vessels and blood stains with sterile hemostatic forceps, discarding waste liquid and flushing;
s3: placing the small umbilical cord section with blood vessels removed into a new culture dish, washing for 3 times until no blood or blood vessels are observed by naked eyes, discarding waste liquid, and cutting the disposable belt into small pieces by using a sterile scissors;
s4: taking out the sterile culture tray, and placing the cut umbilical cord tissue into the culture traySlightly pressing the tissue stack in a sterile culture tray to adhere the umbilical cord tissue to the bottom of the sterile culture tray, placing at 37 ℃ and 5% CO after inoculation2Standing for 4h in a saturated humidity incubator;
s5: after 4h, taking out the sterile culture tray from the incubator, observing whether the umbilical cord tissue is tightly attached to the bottom of the culture tray, adding 10mL of df12+ 10% FBS complete culture solution by using a 10mL pipette after determining that the umbilical cord tissue is tightly attached, not shaking, just submerging the culture solution in a small tissue pile, and not separating the small tissue pile from the bottom of the culture bottle, then placing the culture bottle at 37 ℃ and 5% CO2And continuing culturing in a saturated humidity incubator;
s6: after 48 hours, half-amount liquid is changed for the first time, and then the full-amount liquid is changed every 5d, in the liquid changing process, in order to avoid that the tissue pile is separated from the bottom of the culture bottle, only the old culture solution can be absorbed by a pipette, and the culture solution cannot be directly poured, and when liquid is added, liquid is added along the side surface of the culture bottle;
s7: culturing to about 6-8 days, climbing out a small amount of adherent cells beside a small pile of visible tissues under a microscope, and gradually amplifying the umbilical cord mesenchymal stem cells, wherein the result is shown in figure 1; the small tissue piles of about 10-12 days slowly separate from the bottom of the culture bottle and float in the culture solution, and the umbilical cord mesenchymal stem cells adhere to the wall by 80-90%, and the result is shown in figure 2; then, most floating tissues are discarded after liquid changing, and the cells can be firstly passaged when the cells reach about 80 percent of adherent or more local cell communities are dense.
The stem cells isolated and cultured from fresh umbilical cord were treated as described above and compared with the stem cells isolated from umbilical cord after preservation with the preservation solution of the present invention, and the results are shown in Table 1.
TABLE 1 characteristics of isolated stem cells of umbilical cord
In addition, the osteogenic, fibroblastic, and chondrogenic differentiation abilities of P1 generation of MSCs isolated from the post-preservation umbilical cord were examined, and the results are shown in fig. 3, fig. 4, and fig. 5, respectively.
As can be seen from table 1 and fig. 3 to 5, after the umbilical cord is preserved for 7 days by using the umbilical cord preservation solution of the present invention, sufficient MSC can be separated, and the osteogenic differentiation, fibroblastic differentiation and chondrogenic differentiation abilities of the separated MSC are similar to those of the MSC separated from fresh umbilical cord; the umbilical cord after preservation is similar to the fresh umbilical cord, which shows that the preservation solution of the invention has good preservation effect.
In order to confirm that the cells isolated from the umbilical cord after preservation were indeed MSCs, the expression of CD73+, CD90+, CD34-, CD45-, etc. by the cells was analyzed by flow cytometry, and the results are shown in fig. 6 to 9. As can be seen from the figure, after 1 day and 7 days of storage, the separated cells highly express CD73+ and CD90+, but do not express CD 34-and CD45-, which accords with the immunophenotype of MSC, and the separated cells are MSC.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (5)
1. An umbilical cord preservation solution characterized by comprising, in 1L of preservation solution:
15-20 mmol of hyperbranched polyglycerol, 99-101 mmol of lactobionic acid, 24-26 mmol of monopotassium phosphate, 4-6 mmol of magnesium sulfate, 4-6 mmol of adenosine, 2-4 mmol of glutathione reduced form, 6-10 mg of dexamethasone, 0.5-1.5 mmol of allopurinol, 50-70 mmol of sucrose, 40-60 g of dextran-4040, 18-22 mg of isobodidine, 4-6 ml of sodium hydroxide and 19-21 ml of potassium hydroxide.
2. The umbilical cord preservation solution according to claim 1, comprising, in terms of 1L preservation solution:
18mmol of hyperbranched polyglycerol, 100mmol of lactobionic acid, 25mmol of monopotassium phosphate, 5mmol of magnesium sulfate, 5mmol of adenosine, 3mmol of glutathione reduced form, 8mg of dexamethasone, 1mmol of allopurinol, 60mmol of sucrose, 4050 g of dextran, 20mg of isobinder, 5ml of sodium hydroxide and 20ml of potassium hydroxide.
3. The umbilical cord preservation solution according to claim 1, wherein: the pH value of the preservation solution is 7.45 +/-0.10, and the osmotic pressure is 310 +/-10 mOSM/L.
4. A method for preserving an umbilical cord, comprising the steps of:
s1: taking a newborn umbilical cord, sequentially cleaning the newborn umbilical cord with alcohol and a PBS solution, and dividing the cleaned umbilical cord into small sections with the length of 3-5 cm;
s2: putting the segmented umbilical cord into a storage container, adding the umbilical cord storage solution according to any one of claims 1 to 3 until the umbilical cord is completely submerged, and then placing the storage container in an environment at 3-5 ℃ for storage.
5. The saving method according to claim 4, comprising the steps of: the storage temperature in S2 was 4 ℃.
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Cited By (3)
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| CN112790189A (en) * | 2021-03-10 | 2021-05-14 | 四川大学华西医院 | Organ perfusion and preservation solution and application thereof |
| CN113767895A (en) * | 2021-09-23 | 2021-12-10 | 北京大麦植发技术研究有限公司 | Composite hair follicle tissue preservation solution, preparation method thereof and preservation method for maintaining hair follicle in-vitro activity |
| CN114223651A (en) * | 2021-12-29 | 2022-03-25 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in human renal epithelial cells |
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| CN114223651A (en) * | 2021-12-29 | 2022-03-25 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in human renal epithelial cells |
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Application publication date: 20201211 |