Method for extracting and separating cord blood and placenta stem cells of sika deer

Abstract

The invention discloses a method for extracting and separating cord blood and placenta stem cells of sika deer, and belongs to the technical field of biomedicine. The problems that an existing stem cell extraction and separation method is low in efficiency, poor in cell activity, low in purity and large in pollution risk are solved. The method comprises the following steps: collecting cord blood and placenta after delivery of sika deer; diluting the cord blood, performing density gradient centrifugation, collecting the albuginea layer enriched with the stem cells, adding an erythrocyte lysate to remove residual erythrocytes, and centrifuging and collecting the stem cells; the method comprises the following steps: cutting placenta tissues into pieces, putting the placenta tissues into trypsin-EDTA digestive juice containing collagenase, carrying out ultrasonic-assisted digestion, and centrifuging to obtain stem cells enriched at the bottom of a centrifugal tube; and finally, resuspending the collected stem cells in a culture medium containing fetal calf serum and growth factors, adjusting the cell concentration, and inoculating into a cell culture bottle for culture. According to the method, the extraction efficiency and quality of the sika deer cord blood and placenta stem cells can be remarkably improved, and the activity and safety of the cells are ensured.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for extracting and separating sika deer umbilical cord blood and placental stem cells. Background Technology

[0002] Stem cells are special cells with self-renewal capacity and multi-directional differentiation potential, possessing considerable application potential in regenerative medicine, disease treatment, and anti-aging, and are gradually becoming a core research target in the life sciences. Their most prominent feature is their ability to differentiate into various functional cell types, such as neurons, cardiomyocytes, and hepatocytes. Based on their origin and differentiation potential, stem cells can be broadly classified into embryonic stem cells, adult stem cells, and induced pluripotent stem cells, among others. Recent studies have shown that the placenta and umbilical cord, in addition to containing a large number of blood-derived stem cells, also harbor many adult stem cells from solid tissues, including mesenchymal stem cells, pluripotent adult stem cells, and fibroblast stem cells. Previously, the placenta and umbilical cord were considered waste products after childbirth; however, with proper development and utilization, the stem cells in these discarded resources may provide new solutions for clinical operations and basic research.

[0003] Sika deer are valued for their distinct pharmacological characteristics. Their umbilical cord blood and placental tissue contain abundant stem cell resources. These stem cells possess low immunogenicity, high proliferative capacity, and good stability, showing potential applications in allogeneic transplantation. Related studies show that sika deer umbilical cord blood and placental stem cells have significant advantages in clinical applications due to their lower immune rejection rate. These stem cells contain many bioactive substances that can promote cell proliferation and differentiation, providing a suitable survival environment for the cells. Because of their high reproductive capacity and abundant umbilical cord blood and placental resources, sika deer are considered a primary candidate for large-scale stem cell isolation. While placenta, umbilical cord, and adipose tissue differ significantly in structural characteristics and cellular composition, they are all solid biological materials containing adult stem cells. Although the types and quantities of stem cells vary across these tissues, their cell bank storage methods are common, providing theoretical support for creating a unified technical process.

[0004] Currently, stem cell extraction and separation technologies have significant limitations in terms of efficiency, purity, and cell viability. Low extraction efficiency, reduced cell viability, and insufficient purity are the main problems. Furthermore, there is a risk of exogenous impurities or pathogen contamination. These factors not only affect the stability of stem cell quality but also pose risks to their safety and efficacy in clinical applications. To meet the demand for high-quality stem cells in regenerative medicine and tissue engineering, there is an urgent need to develop new, efficient, safe, and highly pure extraction and separation technologies. Summary of the Invention

[0005] This invention provides a highly efficient, safe, and high-purity method for extracting and separating sika deer umbilical cord blood and placental stem cells, aiming to solve the problems of low extraction efficiency, poor cell viability, low purity, and high risk of contamination in existing sika deer extraction and separation methods. The method of this invention, by optimizing the extraction process and combining it with modern biotechnology, can significantly improve the extraction efficiency and quality of sika deer umbilical cord blood and placental stem cells, while ensuring cell viability and safety.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a method for extracting and separating sika deer umbilical cord blood and placental stem cells, comprising:

[0008] Step 1: After the sika deer gives birth, cut the umbilical cord within 30 seconds, clean the surface of the umbilical cord with disinfectant, locate the umbilical vein puncture point, insert a blood collection needle to draw blood, and let the blood flow naturally into a blood storage bag containing anticoagulant to obtain umbilical cord blood.

[0009] After removing the placenta from the doe, remove the fetal membranes, blood vessels, fat and connective tissue. Under sterile conditions, rinse at least three times with Tris-HCl buffer containing sodium EDTA, antibiotic solution and sodium chloride.

[0010] Step 2: Dilute umbilical cord blood with phosphate-buffered saline (PBS) and add the diluted umbilical cord blood to the upper layer of Ficoll-Paque separation medium. After density gradient centrifugation, discard the supernatant. Stem cells are enriched in the intermediate white membrane layer. Transfer the entire intermediate white membrane layer to a centrifuge tube, add red blood cell lysis buffer, mix well, lyse the red blood cells, centrifuge to discard the red supernatant, wash with PBS, and collect the stem cells.

[0011] The placenta was washed with physiological saline and cut into pieces smaller than 1 mm3. The placental tissue was placed in a trypsin-EDTA digestion solution containing collagenase and sonicated at a frequency of up to 30 kHz. After digestion, the tissue was mechanically broken up to obtain a single-cell suspension. The single-cell suspension was passed through a 100-mesh sieve and the filtrate was collected. The filtrate was added to a centrifuge tube and centrifuged. The supernatant was removed, the cell pellet was collected, and the cell pellet was resuspended in a culture medium. After centrifugation, the supernatant was discarded, and the stem cells were enriched at the bottom of the centrifuge tube.

[0012] Step 3: Stem cells collected from umbilical cord blood and placenta are added to culture medium, mixed, centrifuged, and the supernatant is removed to obtain an enriched stem cell pellet. The enriched stem cell pellet is then resuspended in a culture medium containing fetal bovine serum and growth factors, and the stem cell concentration is adjusted to 3 × 10⁻⁶. 5 Cells per milliliter were inoculated into cell culture flasks and cultured in a cell culture incubator at 37°C and 5% CO2.

[0013] Preferably, in step one, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride buffer solution is 0.5 mol / L and the pH is 7.4.

[0014] Preferably, in step one, the concentration of sodium ethylenediaminetetraacetate in the tris(hydroxymethyl)aminomethane hydrochloride buffer is 10 mmol / L, and the concentration of sodium chloride is 1.0 mol / L.

[0015] Preferably, in step one, the antibiotic solution comprises 100 μg / mL gentamicin, 100 U / mL streptomycin, 100 U / mL penicillin and 2.5 μg / mL amphotericin B, the solvent of the antibiotic solution is ultrapure water, and 10 mL of antibiotic solution is added to every 100 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0016] Preferably, in step two, the volume ratio of phosphate buffer used to dilute umbilical cord blood to umbilical cord blood is 1:1.

[0017] Preferably, in step two, the volume ratio of the diluted umbilical cord blood to the Ficoll-Paque separation solution is 2:1.

[0018] Preferably, in step two, the diluted umbilical cord blood is added to the upper layer of the Ficoll-Paque separation solution at a rate of 3 mL / min.

[0019] Preferably, in step two, the volume of the erythrocyte lysis buffer is 5 times the volume of the intermediate white membrane layer, and the lysis time is 2 minutes.

[0020] Preferably, in step two, the concentration of the trypsin-ethylenediaminetetraacetic acid digest solution is 0.25%.

[0021] Preferably, in step two, the concentration of collagenase II in the trypsin-ethylenediaminetetraacetic acid digestion solution is 0.5%.

[0022] Preferably, in step two, the conditions for ultrasonic digestion are: temperature 37°C and time 30 min.

[0023] Preferably, in step two, the conditions for density gradient centrifugation are: linearly increasing the speed to 1900 rpm within 60 seconds, maintaining it for 25 minutes, and then linearly decreasing the speed to 0 rpm within 180 seconds.

[0024] Preferably, in step two, the centrifugation conditions are 1500 rpm for 10-12 min.

[0025] Preferably, in step three, the centrifugation conditions are 1500 rpm for 10-12 min.

[0026] Preferably, in step three, the growth factors include bFGF, IL-3, and IL-6. In the culture medium containing fetal bovine serum and growth factors, the concentration of fetal bovine serum is 20%, the concentration of bFGF is 4 ng / mL, the concentration of IL-3 is 10 ng / mL, and the concentration of IL-6 is 100 ng / mL.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The present invention discloses a method for extracting and separating sika deer umbilical cord blood and placental stem cells. After sika deer give birth, umbilical cord blood and placenta are collected. The umbilical cord blood is diluted with PBS and added to the upper layer of Ficoll-Paque separation medium for centrifugation. The stem cell-rich white membrane layer is collected, and erythrocyte lysis buffer is added to remove residual erythrocytes. The stem cells are then collected by centrifugation. The placental tissue is minced and placed in a trypsin-EDTA digestion solution containing collagenase. The digestion is assisted by low-intensity ultrasound, followed by separation using a cell homogenizer. The cell pellet is collected by centrifugation after filtration and density gradient centrifugation. Finally, the stem cells are collected, resuspended in a culture medium containing fetal bovine serum and growth factors, and after adjusting the cell concentration, they are seeded into cell culture flasks for culture.

[0029] 1. Significantly improved extraction efficiency: This invention improves the extraction process and combines it with modern biotechnology methods, such as low-intensity ultrasound-assisted digestion and density gradient centrifugation, to achieve highly efficient separation of stem cells from sika deer umbilical cord blood and placenta, thus significantly improving the stem cell extraction efficiency.

[0030] 2. Improved cell activity and quality: This invention improves the conditions of mild processing, namely the enzyme concentration, ultrasound parameters and culture temperature, and creates an environment suitable for cell growth, which greatly reduces the degree of cell damage and ensures that the extracted stem cells have high activity and good proliferative potential, providing a high-quality cell resource guarantee for subsequent cell culture and clinical applications.

[0031] 3. Purity Enhancement Mechanism: This invention combines multi-stage centrifugation and density gradient separation technology to remove a large amount of impurities and non-stem cell-related components, greatly improving the purity of the target stem cell population and effectively reducing the risk of potential cell contamination, providing high-quality, high-purity cell samples to support clinical applications and basic research.

[0032] 4. Safety Improvement Design: This invention follows aseptic operation guidelines in the extraction process and uses a combination of broad-spectrum antibiotics such as gentamicin, streptomycin, penicillin, and amphotericin B with antifungal agents, which can significantly reduce the possibility of potential contamination by pathogenic microorganisms and significantly enhance the safety and quality of the extracted stem cells.

[0033] 5. Abundant sources and low cost: Sika deer have a strong reproductive capacity, and umbilical cord blood and placental resources are abundant and easy to collect. This invention forms a highly efficient stem cell separation technology system, which can give full play to the value of these biological materials, significantly reduce extraction costs, and provide reliable technical support for large-scale production and clinical application.

[0034] 6. Highly low immunogenicity: Sika deer umbilical cord blood and placental stem cells have relatively obvious low immunogenicity characteristics. When performing allogeneic transplantation, the probability of immune rejection is significantly reduced. This characteristic not only provides strong support for clinical applications, but also greatly expands the application scope and practical value of stem cell technology.

[0035] This invention, by improving the extraction process and combining it with modern biotechnology, effectively solves the obvious problems exposed by traditional extraction methods in terms of extraction efficiency, cell viability preservation, product purity, and potential contamination risks. This innovative achievement provides key technical support for the basic research and clinical application of sika deer stem cells. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a schematic flowchart of the extraction and separation method of sika deer umbilical cord blood and placental stem cells according to the present invention.

[0038] Figure 2 The result is obtained by density gradient centrifugation of umbilical cord blood stem cells from Example 1 of the present invention. Detailed Implementation

[0039] To further understand the present invention, preferred embodiments of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0040] like Figure 1 As shown, the method for extracting and separating sika deer umbilical cord blood and placental stem cells of the present invention includes:

[0041] Step 1: Cut the umbilical cord within 30 seconds after the sika deer gives birth, clean the surface of the umbilical cord with disinfectant, locate the umbilical vein puncture point, insert a blood collection needle to draw blood, and let the blood flow naturally into the blood bag containing anticoagulant. Avoid excessive shaking or squeezing to prevent cell rupture and obtain umbilical cord blood.

[0042] After removing the placenta from the doe, remove the fetal membranes, blood vessels (arteries and veins), fat and connective tissue. Under sterile conditions, rinse the placenta at least three times with a tris(hydroxymethyl)aminomethane hydrochloride buffer solution containing sodium EDTA, antibiotic solution and sodium chloride.

[0043] Step 2: Dilute umbilical cord blood with phosphate buffer and slowly add the diluted umbilical cord blood to the top layer of Ficoll-Paque separation medium. After density gradient centrifugation (different components are separated into layers according to density, with red blood cells having the highest density (sinking to the bottom), plasma having the lowest density (top layer), and white blood cells and platelets having intermediate densities), discard the supernatant. Stem cells are enriched in the intermediate white membrane layer. Transfer the entire intermediate white membrane layer to a centrifuge tube, then add red blood cell lysis buffer, gently pipette to mix, perform red blood cell lysis, centrifuge to discard the red supernatant, wash with PBS, and collect the stem cells.

[0044] The placenta was washed with physiological saline and cut into pieces smaller than 1 mm3. The obtained placental tissue was placed in a trypsin-EDTA digestion solution containing collagenase and sonicated at a frequency of up to 30 kHz. After digestion, the placenta was mechanically broken up using a handheld cell homogenizer to release single cells and obtain a single-cell suspension. The single-cell suspension was passed through a 100-mesh sieve to remove undigested placental tissue and the filtrate was collected. The filtrate was centrifuged in a centrifuge tube, the supernatant was removed, the cell pellet was collected, and the cell pellet was resuspended in a culture medium. After centrifugation, the supernatant was discarded, and the stem cells were enriched at the bottom of the centrifuge tube.

[0045] Step 3: Add stem cells collected from umbilical cord blood and placenta to culture medium, mix well, centrifuge again, remove the supernatant, and obtain enriched stem cell pellet; then resuspend the enriched stem cell pellet in culture medium containing fetal bovine serum and growth factors, adjusting the stem cell concentration to 3 × 10⁻⁶. 5 Cells per milliliter were inoculated into cell culture flasks and cultured in a cell culture incubator at 37°C and 5% CO2.

[0046] In step one of this invention, the disinfectant is a commonly used reagent in the art and is not particularly limited. For example, in some embodiments, alcohol-based disinfectants are used. The anticoagulant is also a commonly used reagent in the art and is not particularly limited, as long as it can achieve the effect of preventing blood clotting.

[0047] In step one of this invention, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride buffer is 0.5 mol / L, and the pH is 7.4. The antibiotic solution includes 100 μg / mL gentamicin, 100 U / mL streptomycin, 100 U / mL penicillin, and 2.5 μg / mL amphotericin B. The solvent for the antibiotic solution is ultrapure water, and 10 mL of antibiotic solution is added to every 100 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer. In the tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, the concentration of sodium ethylenediaminetetraacetate is 10 mmol / L, and the concentration of sodium chloride is 1.0 mol / L.

[0048] In step one of this invention, the preferred volume ratio of phosphate buffer to umbilical cord blood for diluting is 1:1; the preferred volume ratio of diluted umbilical cord blood to Ficoll-Paque separation solution is 2:1. Preferably, the diluted umbilical cord blood is added to the upper layer of the Ficoll-Paque separation solution at a rate of 3 mL / min.

[0049] In step two of this invention, the concentration of the trypsin-EDTA digestion solution is 0.25%; and the concentration of collagenase II in the trypsin-EDTA digestion solution is 0.5%.

[0050] In step two of this invention, the conditions for ultrasonic digestion are: temperature 37°C and time 30 min.

[0051] In step two of this invention, the conditions for density gradient centrifugation are: linearly increasing the speed to 1900 rpm within 60 s, maintaining it for 25 min, and then linearly decreasing the speed to 0 rpm within 180 s.

[0052] In step two of this invention, the centrifugation conditions are 1500 rpm for 10-12 min.

[0053] In step three of this invention, the centrifugation conditions are 1500 rpm for 10-12 min. The growth factors include bFGF, IL-3, and IL-6. In the culture medium containing fetal bovine serum and growth factors, the concentration of fetal bovine serum is 20%, the concentration of bFGF is 4 ng / mL, the concentration of IL-3 is 10 ng / mL, and the concentration of IL-6 is 100 ng / mL.

[0054] It should be noted that there are no special limitations on the culture medium of the present invention.

[0055] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments.

[0056] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, equipment, etc., used in the following embodiments are commercially available. The sika deer used in the experiments were from Shuangyang District, Changchun City, Jilin Province.

[0057] Example 1

[0058] Methods for the extraction and separation of sika deer umbilical cord blood and placental stem cells include:

[0059] Step 1: Cut the umbilical cord within 30 seconds after the sika deer gives birth, clean the surface of the umbilical cord with disinfectant, locate the umbilical vein puncture point, insert a blood collection needle to draw blood, and let the blood flow naturally into the blood bag containing anticoagulant. Avoid excessive shaking or squeezing to prevent cell rupture and obtain umbilical cord blood.

[0060] After removing the placenta from the doe, the fetal membranes, blood vessels, fat, and connective tissue are removed. Under sterile conditions, the placenta is rinsed at least three times with a Tris-HCl buffer containing sodium EDTA, antibiotic solution, and sodium chloride. The Tris-HCl buffer has a concentration of 0.5 mol / L and a pH of 7.4. The Tris-HCl buffer contains 10 mmol / L sodium EDTA, 1.0 mol / L sodium chloride, and 10 μg / mL gentamicin, 100 U / mL streptomycin, 100 U / mL penicillin, and 2.5 μg / mL amphotericin B. The antibiotic solution is in the form of ultrapure water. 10 mL of antibiotic solution is added to every 100 mL of Tris-HCl buffer.

[0061] Step 2: Dilute the umbilical cord blood with PBS at a volume ratio of 1:1. Add the diluted umbilical cord blood slowly (3 mL / min) to the upper layer of the Ficoll-Paque separation medium at a volume ratio of 2:1. Place the mixture in a low-speed benchtop centrifuge and linearly increase the speed to 1900 rpm over 60 seconds, maintain this speed for 25 minutes, and then linearly decrease the speed to 0 rpm over 180 seconds. After centrifugation, discard the supernatant using a pipette. Stem cells are enriched in the intermediate white membrane layer, as shown below. Figure 2 As shown ( Figure 2 (The mononuclear cell layer in the middle) The entire middle white membrane layer was aspirated into a centrifuge tube, then red blood cell lysis buffer was added, and the mixture was gently blown to mix. Red blood cells were lysed, centrifuged to discard the red supernatant, and the stem cells were collected after washing with PBS.

[0062] The placenta was washed with physiological saline and cut into pieces smaller than 1 mm3. The placental tissue was placed in a sterile container for later use. The placental tissue was then placed in a trypsin-EDTA digestion solution containing 0.5% collagenase. The concentration of the trypsin-EDTA digestion solution was 0.25%. The tissue was sonicated at 30 kHz at 37°C for 30 minutes. After digestion, the tissue was mechanically broken up using a handheld cell homogenizer until single cells were released, resulting in a single-cell suspension.

[0063] The single-cell suspension was passed through a 100-mesh sieve to remove undigested placental tissue. The filtrate was collected and centrifuged at 1500 rpm for 12 min. The supernatant was removed, the cell pellet was collected, and the cell pellet was resuspended in culture medium. After centrifugation at 1500 rpm for 12 min, the supernatant was discarded, and the stem cells were enriched at the bottom of the centrifuge tube.

[0064] Step 3: Centrifuge the stem cells collected from umbilical cord blood and placenta at 1500 rpm for 12 min, remove the supernatant, and obtain an enriched stem cell pellet; then resuspend the enriched stem cell pellet in a culture medium containing fetal bovine serum and growth factors, adjusting the stem cell concentration to 3 × 10⁻⁶. 5 Cells / mL were seeded into cell culture flasks and cultured in a cell culture incubator at 37°C and 5% CO2.

[0065] The growth factors included bFGF, IL-3, and IL-6. In the culture medium containing fetal bovine serum and growth factors, the concentration of fetal bovine serum was 20% v / v, the concentration of bFGF was 4 ng / mL, the concentration of IL-3 was 10 ng / mL, and the concentration of IL-6 was 100 ng / mL.

[0066] Comparative Example 1

[0067] The experimental conditions and procedures were the same as in Example 1, except that in step two, 30kHz ultrasonic digestion was not used. Everything else remained the same.

[0068] In Comparative Example 1, when placental tissue was digested, the use of ultrasound-assisted technology was excluded. The results showed that this method significantly reduced the cell release efficiency, and some tissue samples could not achieve effective dispersion, which in turn led to a significant reduction in the total cell recovery. In this process, mechanical damage also inhibited cell activity and affected its proliferation capacity in subsequent culture.

[0069] Comparative Example 2

[0070] The experimental conditions and steps were the same as in Example 1, except that density gradient centrifugation was not used in step two, while the rest remained the same.

[0071] In Comparative Example 2, during the processing of umbilical cord blood and placental tissue, the Ficoll-Paque density gradient centrifugation method was not used. Instead, differential centrifugation was used to process cells, which significantly reduced cell purity and increased the number of impurities and debris in the separation solution. This greatly affected the enrichment effect of stem cells, and the stability and consistency of cell population quality may also decrease significantly during subsequent culture.

[0072] Comparative Example 3

[0073] The experimental conditions and procedures were the same as in Example 1, except that in step one, the placenta was rinsed with physiological saline instead of repeatedly washing with a buffer solution containing sodium ethylenediaminetetraacetate, antibiotic solution, and sodium chloride. Everything else remained the same.

[0074] In Comparative Example 3, when cleaning the placenta, only physiological saline was used for cleaning without adding sodium EDTA, antibiotic solution, or Tris-HCl buffer. It was found that it was difficult to effectively remove the substances remaining on the surface of the placental tissue. This resulted in a very high load of foreign matter and microorganisms, and greatly increased the possibility of cell contamination. This could easily lead to bacterial or fungal infections during the subsequent culture process.

[0075] Comparative Example 4

[0076] The experimental conditions and procedures were the same as in Example 1, except that in step two, a handheld cell homogenizer was not used to mechanically break down the cells to release single cells. Everything else remained the same.

[0077] In Comparative Example 4, after digesting placental tissue, cells were released solely by the digestion fluid without mechanical separation using a handheld cell homogenizer. This resulted in larger tissue blocks, incomplete cell release, and lower cell yield. Poor cell dispersion also negatively impacted subsequent centrifugation and enrichment efficiency.

[0078] Comparative Example 5

[0079] The experimental conditions and procedures were the same as in Example 1, except that in step two, instead of linearly increasing the speed to 1900 rpm within 60 seconds, maintaining it for 25 minutes, and then linearly decreasing it to 0 rpm within 180 seconds, the centrifugation was fixed at 1900 rpm for 25 minutes. Everything else remained the same.

[0080] In Comparative Example 5, during the centrifugation of umbilical cord blood and placental tissue, no acceleration or deceleration time was set. The centrifugation speed was directly set to 1900 rpm and run continuously for 25 minutes. It was found that the cells were subjected to greater shear stress during centrifugation, which caused some cells to be damaged and their activity to decrease. Such uneven cell sedimentation distribution will have a great impact on the subsequent stem cell separation and enrichment effect.

[0081] The cell proliferation rate of Example 1 and Comparative Examples 1-5 was detected by adding 2×10⁻⁶ cells to each well of a 96-well plate. 3 Cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cells were then removed and their OD values ​​were measured at 450 nm using a microplate reader. The cells were shaken to mix thoroughly before measurement. The results are shown in Table 1.

[0082] Table 1. Results of cell proliferation rate detection in Example 1 and Comparative Examples 1-5

[0083] Group Cell proliferation rate Example 1 185±5.64％ Comparative Example 1 161±5.21％ Comparative Example 2 153±4.32％ Comparative Example 3 151±4.68％ Comparative Example 4 143±3.59％ Comparative Example 5 138±3.11％

[0084] As can be seen from the above comparison, the key methods used in Example 1 of this invention, such as ultrasonic-assisted digestion, density gradient centrifugation, optimized cleaning solution formulation design, and mechanical separation, not only improve the efficiency and purity of stem cell extraction, but also enhance their biological activity. At the same time, they reduce the potential contamination of stem cells. The improved centrifugation process can better ensure the survival of cells during extraction and greatly reduce damage to cells.

[0085] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the embodiments. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all embodiments here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting and separating sika deer umbilical cord blood and placental stem cells, characterized in that, include: Step 1: After the sika deer gives birth, cut the umbilical cord within 30 seconds, clean the surface of the umbilical cord with disinfectant, locate the umbilical vein puncture point, insert a blood collection needle to draw blood, and let the blood flow naturally into a blood storage bag containing anticoagulant to obtain umbilical cord blood. After removing the placenta from the doe, remove the fetal membranes, blood vessels, fat and connective tissue. Under sterile conditions, rinse at least three times with a tris(hydroxymethyl)aminomethane hydrochloride buffer solution containing sodium EDTA, antibiotic solution and sodium chloride, and set aside for use. Step 2: Dilute umbilical cord blood with phosphate buffer and add the diluted umbilical cord blood to the upper layer of Ficoll-Paque separation medium. After density gradient centrifugation, discard the supernatant. Stem cells are enriched in the intermediate white membrane layer. Transfer the entire intermediate white membrane layer to a centrifuge tube, add red blood cell lysis buffer, mix well, lyse the red blood cells, centrifuge and discard the red supernatant. After washing with phosphate buffer, collect the stem cells. The placenta was washed with physiological saline and cut into pieces smaller than 1 mm3. The placental tissue was placed in a trypsin-EDTA digestion solution containing collagenase and sonicated at a frequency of up to 30 kHz. After digestion, the tissue was mechanically broken up to obtain a single-cell suspension. The single-cell suspension was passed through a 100-mesh sieve and the filtrate was collected. The filtrate was added to a centrifuge tube and centrifuged. The supernatant was removed, the cell pellet was collected, and the cell pellet was resuspended in a culture medium. After centrifugation, the supernatant was discarded, and the stem cells were enriched at the bottom of the centrifuge tube. Step 3: Stem cells collected from umbilical cord blood and placenta are added to culture medium, mixed, centrifuged, and the supernatant is removed to obtain an enriched stem cell pellet. The enriched stem cell pellet is then resuspended in a culture medium containing fetal bovine serum and growth factors, and the stem cell concentration is adjusted to 3 × 10⁻⁶. 5 Cells per milliliter were inoculated into cell culture flasks and cultured in a cell culture incubator at 37°C and 5% CO2.

2. The method for extraction and separation of sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step one, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride buffer solution is 0.5 mol / L, and the pH is 7.

4. In the tris(hydroxymethyl)aminomethane hydrochloride buffer solution, the concentration of sodium ethylenediaminetetraacetate is 10 mmol / L and the concentration of sodium chloride is 1.0 mol / L. The antibiotic solution comprises 100 μg / mL gentamicin, 100 U / mL streptomycin, 100 U / mL penicillin and 2.5 μg / mL amphotericin B. The solvent for the antibiotic solution is ultrapure water, and 10 mL of antibiotic solution is added to every 100 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer.

3. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step two, the volume ratio of phosphate buffer used to dilute umbilical cord blood to umbilical cord blood is 1:1, and the volume ratio of the diluted umbilical cord blood to Ficoll-Paque separation solution is 2:

1.

4. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step two, the diluted umbilical cord blood is added to the upper layer of the Ficoll-Paque separation solution at a rate of 3 mL / min.

5. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step two, the volume of the red blood cell lysis buffer is five times the volume of the intermediate white membrane layer, and the lysis time is 2 minutes.

6. The method for extraction and separation of sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step two, the concentration of the trypsin-ethylenediaminetetraacetic acid digestion solution is 0.25%, and the concentration of collagenase II in the trypsin-ethylenediaminetetraacetic acid digestion solution is 0.5%.

7. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step two, the conditions for ultrasonic digestion are: temperature 37°C and time 30 minutes.

8. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step two, the conditions for density gradient centrifugation are: linearly increasing the speed to 1900 rpm within 60 seconds, maintaining it for 25 minutes, and then linearly decreasing the speed to 0 rpm within 180 seconds. The centrifugation conditions were all 1500 rpm for 10-12 min.

9. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step three, the centrifugation conditions are 1500 rpm for 10-12 min.

10. The method for extracting and separating sika deer umbilical cord blood and placental stem cells according to claim 1, characterized in that, In step three, the growth factors include bFGF, IL-3, and IL-6. In the culture medium containing fetal bovine serum and growth factors, the concentration of fetal bovine serum is 20%, the concentration of bFGF is 4 ng / mL, the concentration of IL-3 is 10 ng / mL, and the concentration of IL-6 is 100 ng / mL.

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