Preserving fluid for decidua basalis tissues as well as preparation method and use method of preserving fluid

Abstract

The invention belongs to the technical field of tissue preservation, and particularly relates to a preserving fluid for decidua basalis tissues as well as a preparation method and a use method of the preserving fluid. The preserving fluid of the decidua basalis tissue is an alpha-MEM culture medium containing 1-2g / mL of trehalose, 0.5-0.8 mol / L of ammonium chloride, 1-2mmol / L of sodium pyruvate, 10 <-8 >-2 * 10 <-8 > mol / L of estrogen, 10 <-8 >-2 * 10 <-8 > mol / L of progestational hormone, 0.5-0.8 mmol / L of adenosine and 10-15mu g / mL of tetracycline hydrochloride. According to the preserving fluid for the decidua basalis tissue, multiple components are scientifically added in proportion, red blood cells can be effectively removed in the tissue pretreatment process, and the activity and yield of primary stem cells of the decidua basalis are improved; the preserving fluid can preserve decidua basalis tissues for a long time, and the tissue activity is not affected within 48 hours.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue preservation, and in particular relates to a preservation solution for decidua basalis tissue and a preparation method and a use method thereof. Background Art

[0002] Decidua basalis (DB) is a special tissue layer formed by the endometrium. It forms the bottom of the placenta, provides structural and vascular support for the fetus and placenta, and also plays an important role in immunoregulation.

[0003] Decidua basal tissue contains a large number of decidua basal stem cells. Decidua basalismesenchymal stem cells (DB-MSCs) are stem cells with multidirectional differentiation potential isolated from human placenta decidua basal tissue. As a type of perinatal stem cell, DB-MSCs have the potential to differentiate into three germ layers, and have the advantages of sufficient source, low immunogenicity, non-invasive acquisition process, and no social ethical controversy. At present, DB-MSCs have been widely used in preclinical studies of various diseases, including graft-versus-host disease, recurrent miscarriage, hemorrhagic cystitis, allogeneic hematopoietic cell transplantation and acute respiratory distress syndrome, all of which have shown good therapeutic effects and safety.

[0004] After the decidua basalis tissue is separated from the in vivo environment, the cells are easily disturbed by a variety of external factors, and the oxidative stress level increases significantly, causing cell damage. In addition, when the cells lack nutrition and energy, the intracellular energy metabolism will be unbalanced, and the cells will undergo autophagy. When the accumulation rate of damaged cells exceeds the clearance capacity of cell autophagy, the cell capacity is over-consumed, the ATP level drops rapidly, and the cell activity decreases or even dies. At the same time, the decidua basalis tissue is connected to a large number of blood vessels, and blood cells seriously affect the activity and yield of primary cells during the subsequent extraction of decidua basalis stem cells. Therefore, maintaining the activity of the decidua basalis tissue and removing blood cells directly affect the isolation and culture of decidua basalis stem cells and subsequent cell therapy applications, and the preservation of the decidua basalis tissue is particularly important.

[0005] The disadvantages of the prior art are:

[0006] Existing tissue preservation fluids are mainly composed of basal culture medium and other nutrients and antibacterial ingredients. Although these measures can maintain the activity of tissues to a certain extent, the nutrients are single and for decidua basalis tissue, red blood cells cannot be effectively removed, affecting the activity and yield of subsequent extracted primary cells.

[0007] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0008] The inventors of the present invention have found through research that the chorionic plate emits about 60 villi and extends into the decidua basalis. The villi are soaked with blood, so too many red blood cells will be mixed in during separation, resulting in reduced stem cell activity and a longer proliferation cycle. The key to extracting decidua basalis stem cells is to increase the number of stem cells, ensure the survival rate of stem cells, and reduce the proliferation cycle of primary cells as much as possible.

[0009] Therefore, the present method provides a basal decidua tissue preservation solution, which has simple and clear ingredients, is safe and has no toxic side effects, can well maintain the cell viability of the basal decidua tissue after ex vivo, and can also effectively remove red blood cells during tissue pretreatment, thereby improving the activity and yield of basal decidua primary stem cells.

[0010] The solution of the present invention provides a preservation solution for decidua basalis tissue, wherein the preservation solution for decidua basalis tissue is an α-MEM culture medium comprising trehalose, ammonium chloride, sodium pyruvate, estrogen, progesterone, adenosine, and tetracycline hydrochloride.

[0011] Preferably, the preservation solution of the decidua basalis tissue comprises 1-2 g / mL trehalose, 0.5-0.8 mol / L ammonium chloride, 1-2 mmol / L sodium pyruvate, 10 -8 ~2×10 -8 mol / L estrogen, 10 -8 ~2×10 -8 α-MEM culture medium containing 10 mol / L progesterone, 0.5-0.8 mmol / L adenosine, and 10-15 μg / mL tetracycline hydrochloride.

[0012] Preferably, the preservation solution of the decidua basalis tissue comprises 1 g / mL trehalose, 0.5 mol / L ammonium chloride, 1 mmol / L sodium pyruvate, 10 -8 mol / L estrogen, 10 -8 α-MEM medium containing 10 μg / mL tetracycline hydrochloride and 0.5 mmol / L progesterone.

[0013] In order to facilitate the understanding of the present invention, the relevant components of the present invention are described:

[0014] Trehalose: It is a non-permeable preservative that can stabilize the structure of biological membranes and proteins, protect organisms from damage caused by oxidative stress, and has the effects of resisting dehydration, freezing, and hypertonicity.

[0015] Ammonium chloride: can cause changes in osmotic pressure and cause red blood cells to rupture.

[0016] Sodium pyruvate: It is an intermediate product in glycolysis. It can directly enter and exit cells, provide energy for cells, and is also a carbon source required for anabolism.

[0017] Estrogen and progesterone: The two synergistically regulate apoptosis by acting on mitochondria.

[0018] Adenosine: It is an endogenous nucleoside. It is a precursor of ATP and also a metabolite of ATP. It can protect cells by maintaining intracellular ATP levels.

[0019] Tetracycline hydrochloride: Salt is a broad-spectrum polyketide antibiotic with activity against a wide range of Gram-positive and Gram-negative bacteria, atypical organisms such as Chlamydia, Mycoplasma and Rickettsia, and protozoan parasites.

[0020] Based on the same technical concept, the present invention provides a method for preparing the preservation solution of the decidua basalis tissue, and the preparation method is: adding trehalose, ammonium chloride, sodium pyruvate, estrogen, progesterone, adenosine, and tetracycline hydrochloride to α-MEM culture medium and stirring evenly.

[0021] Preferably, the stirring time is 5 to 10 minutes.

[0022] Based on the same technical concept, the present invention provides a method for using the preservation solution of the decidua basalis tissue, and the method for using is:

[0023] (1) Clean the placenta after delivery and obtain the basal decidua tissue;

[0024] (2) cutting the decidua basalis tissue into pieces;

[0025] (3) The chopped chorionic tissue is stored in the storage solution of the basal decidua tissue.

[0026] Preferably, in step (2), the decidua basalis tissue is cut into pieces of 3 to 5 mm. 3 .

[0027] Preferably, in step (3), the storage time does not exceed 48 hours.

[0028] The beneficial effects of the present invention are:

[0029] The preservation solution for decidua basalis tissue of the present invention can effectively remove red blood cells during tissue pretreatment and improve the activity and yield of decidua basalis primary stem cells by adding multiple ingredients in proportion; the preservation solution can preserve decidua basalis tissue for a long time and does not affect tissue activity within 48 hours.

[0030] The preparation method of the present invention only requires adding each component to the α-MEM culture medium, and the operation is simple and easy to control. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is an Alizarin Red staining picture.

[0033] Figure 2 This is an Oil Red O staining picture.

[0034] Figure 3 This is an Alcian blue staining image. DETAILED DESCRIPTION

[0035] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0036] Example 1

[0037] This embodiment provides a preservation solution for basal decidua tissue, which comprises 1 g / mL trehalose, 0.5 mol / L ammonium chloride, 1 mmol / L sodium pyruvate, 10 -8 mol / L estrogen, 10 -8 α-MEM medium containing 10 μg / mL tetracycline hydrochloride and 0.5 mmol / L progesterone.

[0038] Example 2

[0039] This embodiment provides a preservation solution for decidua basalis tissue, which comprises 1 g / mL trehalose, 0.3 mol / L ammonium chloride, 1 mmol / L sodium pyruvate, 10 -8 mol / L estrogen, 10 -8 α-MEM medium containing 10 μg / mL tetracycline hydrochloride and 0.5 mmol / L progesterone.

[0040] Comparative Example 1

[0041] This comparative example provides a similar preservation solution, which is an α-MEM medium containing 1 g / mL trehalose, 1 mmol / L sodium pyruvate, and 10 μg / mL tetracycline hydrochloride.

[0042] Comparative Example 2

[0043] This comparative example provides a similar storage solution, which is an α-MEM medium containing 10 μg / mL tetracycline hydrochloride.

[0044] Effect verification example

[0045] (I) Isolation and preservation of basal decidua tissue

[0046] The placenta after delivery was cleaned with physiological saline, and the basal decidua tissue was cut with surgical forceps and surgical scissors and cut into 3-5 mm2 tissue. The tissue was divided into 8 equal parts and stored in 4 kinds of basal decidua tissue preservation solutions (Example 1, Example 2, Comparative Example 1, Comparative Example 2) for 24h and 48h respectively.

[0047] (II) Isolation of Decidua Basal Stem Cells

[0048] Transfer the tissue mixture to a 50mL centrifuge tube, centrifuge at 500rpm for 5min, discard the supernatant, and take the basal decidua tissue and place it in a 50mL centrifuge tube. Add 100μg / mL type I collagenase (tissue volume: collagenase volume = 1:2) to the centrifuge tube, place it in a shaker at 37℃ and 200rpm, and add serum substitute after 30min to terminate digestion. Centrifuge the tissue mixture at 1000rpm for 5min, discard the supernatant, add serum-free stem cell culture medium and inoculate it into a T25 culture flask, place it in a 37℃, 5% CO2 incubator, and observe the cell growth.

[0049] (III) Experimental results

[0050] 1. Detection of cell proliferation, number and viability is shown in Table 1.

[0051] Table 1 Comparison of time for cells to reach 80% confluence

[0052]

[0053] The P0 generation decidua basalis stem cells were taken, washed once with saline, added with 0.25% trypsin and incubated for 2-3 min, and serum substitute was added to terminate the digestion. 10 μL of the digested decidua basalis stem cell suspension was taken, and 10 μL of 0.4% trypan blue dye was added. The mixture was mixed evenly and added to a cell counting plate. Countstar was used to detect the cell viability. The results are shown in Table 2.

[0054] Table 2 Comparison of cell number and viability

[0055]

[0056]

[0057] From the results in Tables 1 and 2, it can be seen that compared with Comparative Example 1, Comparative Example 2 and Example 2, the cell growth rate, number and viability of Example 1 are significantly higher than those of the other three groups, and there is no significant difference in the number and viability of cells at 24h and 48h in Example 1. It can be concluded that the basal decidua tissue preservation solution described in Example 1 can extend the storage time of basal decidua tissue to 48h. The cell growth rate and number of Example 2 are slightly lower than those of Example 1, but higher than those of Examples 3 and 4. It can be concluded that ammonium chloride can effectively reduce the influence of blood cells on primary cell extraction, thereby increasing the yield of primary cells.

[0058] 2. Identification of cell surface markers

[0059] The P3 generation decidual stem cells were taken, washed once with saline, incubated for 2-3 min with 0.25% trypsin, and digested by adding serum substitute. The digested decidual stem cell suspension was taken, and CD73-APC, CD90-FITC, CD105-PerCP and CD34 / 14 / 19 / 45 / HLA-DR antibodies were added respectively and mixed, and incubated at room temperature in the dark for 15 min. After staining, PBS was added to wash the cells, centrifuged at 300 g for 5 min, the supernatant was discarded, PBS was added to resuspend, and the cells were detected by flow cytometry. The results are shown in Table 3.

[0060] Table 3 Comparison of cell flow cytometry identification

[0061]

[0062]

[0063] From the results in Table 3, it can be seen that compared with Comparative Example 1, Comparative Example 2 and Example 2, the cells separated in Example 1 have a higher purity and meet the characteristics of mesenchymal stem cells. The expression levels of CD73, CD105, and CD90 are higher than 97%, and the expression levels of CD34 / 14 / 19 / 45 / HLA-DR are lower than 2%. At the same time, there is no significant difference between 24h and 48h in Example 1. It can be concluded that the decidua basalis stem cell tissue preservation solution described in Example 1 can improve the purity of the obtained primary cells and extend the storage time of the decidua basalis tissue to 48h. The cell growth rate and number of Example 2 are slightly lower than those of Example 1, but higher than those of Example 3 and Example 4. It can be concluded that ammonium chloride can effectively reduce the influence of blood cells on the extraction of primary cells, thereby improving the purity of decidua basalis stem cells.

[0064] 3. Identification of cell differentiation ability

[0065] The P4 generation decidua basalis stem cells of Example 1 were taken, washed once with physiological saline, incubated for 2-3 min with 0.25% trypsin, and terminated by adding serum substitute. The digested decidua basalis stem cell suspension was taken, and the appropriate cell density was adjusted to be inoculated into a culture dish, and cultured in a 37°C, 5% CO2 incubator until the confluence was 70-80%.

[0066] (1) Remove the old culture medium and add osteogenic differentiation medium. Replace the osteogenic differentiation medium every 3 days. After culturing in a 37°C, 5% CO2 incubator for 21 days, remove the old culture medium, add PBS to wash the cells once, add 4% paraformaldehyde solution to fix at room temperature for 30 minutes, remove the solution, and add PBS to wash the cells twice. Add Alizarin Red dye for 5 minutes, remove the solution, add PBS to wash the cells twice, and observe the staining effect under a microscope. Figure 1 shown. Figure 1 Obvious red-stained mineralized nodule deposition can be observed, indicating that the decidua basalis stem cells extracted in Example 1 have the ability to differentiate into osteoblasts.

[0067] (2) Remove the old culture medium and add adipogenic differentiation medium. Replace the adipogenic differentiation medium every 3 days. After culturing in a 37°C, 5% CO2 incubator for 21 days, remove the old culture medium, add PBS to wash the cells once, add 4% paraformaldehyde solution to fix at room temperature for 30 minutes, remove the solution, and add PBS to wash the cells twice. Add Oil Red O staining solution for 30 minutes, remove the solution, add PBS to wash the cells twice, and observe the staining effect under a microscope. Figure 2 shown. Figure 2 Orange-red lipid droplets can be observed, indicating that the decidua basalis stem cells extracted in Example 1 have the ability to differentiate into adipocytes.

[0068] (3) Remove the old culture medium and add chondrogenic differentiation medium. Replace the culture medium with chondrogenic differentiation medium every 3 days. After culturing in a 37°C, 5% CO2 incubator for 28 days, remove the old culture medium, add PBS to wash the cells once, add 4% paraformaldehyde solution to fix at room temperature for 30 minutes, remove the solution, and add PBS to wash the cells twice. Add Alcian blue dye for 30 minutes, remove the solution, and add PBS to wash the cells twice. Observe the staining effect under a microscope. Figure 3 shown. Figure 3 Acidic mucopolysaccharide stained in blue was observed, indicating that the decidua basalis stem cells extracted in Example 1 had the ability to differentiate into chondrogenic cartilage.

[0069] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A preservation solution for decidua basalis tissue, characterized in that: The preservation liquid of the decidua basalis tissue is an α-MEM culture medium including trehalose, ammonium chloride, sodium pyruvate, estrogen, progesterone, adenosine, and tetracycline hydrochloride.

2. The preservation solution for decidua basalis tissue according to claim 1, characterized in that: The preservation solution of the decidua basalis tissue comprises 1-2 g / mL trehalose, 0.5-0.8 mol / L ammonium chloride, 1-2 mmol / L sodium pyruvate, 10 -8 ~2×10 -8 mol / L estrogen, 10 -8 ~2×10 -8 α-MEM culture medium containing 10 mol / L progesterone, 0.5-0.8 mmol / L adenosine, and 10-15 μg / mL tetracycline hydrochloride.

3. The preservation solution for decidua basalis tissue according to claim 1, characterized in that: The preservation solution of the decidua basalis tissue includes 1g / mL trehalose, 0.5mol / L ammonium chloride, 1mmol / L sodium pyruvate, 10 -8 mol / L estrogen, 10 -8 α-MEM medium containing 10 μg / mL tetracycline hydrochloride and 0.5 mmol / L progesterone.

4. The method for preparing the preservation solution for decidua basalis tissue according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: adding trehalose, ammonium chloride, sodium pyruvate, estrogen, progesterone, adenosine and tetracycline hydrochloride into α-MEM culture medium and stirring the mixture evenly.

5. The method for preparing the preservation solution of decidua basalis tissue according to claim 4, characterized in that: The stirring time is 5 to 10 minutes.

6. The method for using the storage solution for decidua basalis tissue according to any one of claims 1 to 3, characterized in that: The method of use is: (1) Clean the placenta after delivery and obtain the basal decidua tissue; (2) cutting the decidua basalis tissue into pieces; (3) The chopped chorionic tissue is stored in the storage solution of the basal decidua tissue.

7. The method for using the preservation solution for decidua basalis tissue according to claim 6, characterized in that: In step (2), the decidua basalis tissue is cut into pieces of 3 to 5 mm. 3 .

8. The method for using the preservation solution for decidua basalis tissue according to claim 6, characterized in that: In step (3), the storage time does not exceed 48 hours.

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