CN118006549A - Stem cell collection method with small damage to stem cells - Google Patents
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Abstract
The invention relates to the technical field of stem cell collection, in particular to a stem cell collection method with small damage to stem cells, which comprises the following steps: s1: collecting a biological sample containing stem cells; s2: placing the sample obtained in the step S1 into a special buffer solution; s3: a temperature-controlled centrifugation process by gradually increasing the temperature; s4: separating the stem cells from other cell types; s5: transferring the stem cells separated in the step S4 into a microenvironment containing specific nutritional factors and growth factors; s6: applying a low-intensity pulsed electric field technique in a culture environment; s7: the collection was performed using cell biomarkers and high resolution microscopy imaging techniques. The invention remarkably improves the quality and proliferation efficiency of stem cells through mild non-enzymatic separation, optimized culture conditions and real-time quality monitoring, simultaneously maintains the bioactivity of the stem cells, and provides high-quality stem cells for biomedical research and clinical application.
Description
Technical Field
The invention relates to the technical field of stem cell collection, in particular to a stem cell collection method with small damage to stem cells.
Background
In modern biomedical research and clinical applications, the collection and utilization of stem cells has become an important field, and stem cells, particularly stem cells derived from bone marrow or umbilical cord blood, have great potential in regenerative medicine, disease treatment, tissue engineering, etc. because of their ability to self-renew and multi-directionally differentiate, however, the efficient and safe collection and culture of these cells has been a technical challenge facing the field.
The current methods for collecting and processing stem cells have a plurality of limitations, firstly, the traditional cell separation technology often relies on enzymolysis separation or mechanical separation, the methods may damage the biological activity and the integrity of the stem cells, secondly, the stem cells are easy to differentiate or lose activity in the in vitro culture process, which limits the clinical application potential of the stem cells, and furthermore, the current stem cell culture technology is often tedious and low in efficiency, which prevents the wide application of the stem cells to a certain extent.
Disclosure of Invention
Based on the above objects, the present invention provides a stem cell collection method with little damage to stem cells.
A stem cell collection method with small damage to stem cells, comprising the following steps:
S1: collecting a biological sample containing stem cells from bone marrow or umbilical cord blood, and performing physical separation by low-speed centrifugation;
s2: placing the sample obtained in the step S1 into a special buffer solution, and treating the biological sample to separate stem cells;
S3: optimizing the collection efficiency of stem cells by gradually increasing the temperature through a temperature-controlled centrifugation process;
s4: separating stem cells from other cell types by adopting a differential sedimentation method;
s5: transferring the stem cells isolated in S4 into a microenvironment containing specific trophic factors and growth factors to promote proliferation and maturation thereof;
s6: applying a low-intensity pulsed electric field technique in a culture environment to enhance the bioactivity and proliferation capacity of stem cells;
s7: and the quality and the number of the stem cells are monitored in real time by adopting a cell biomarker and a high-resolution microscopic imaging technology, so that the stem cells are effectively collected.
Further, the physical separation in S1 by low-speed centrifugation specifically includes:
S11: transferring the collected bone marrow sample or umbilical cord blood sample into a sterile container, extracting the sample with a volume of 5-10 ml, and adding an equal volume of anticoagulant to prevent blood coagulation;
S12: placing the container containing the sample in a centrifuge under aseptic condition, and setting the centrifugation speed to be 200-250g and the centrifugation time to be 10-15 minutes;
s13: after centrifugation is completed, the upper plasma and middle leukocytes are removed, leaving a bottom pellet containing stem cells.
Further, the step S2 specifically includes:
S21: transferring the bottom sediment containing the stem cells obtained in the step S1 to a new sterile container;
S22: adding a special buffer solution into a sterile container, wherein the buffer solution consists of phosphate buffer physiological saline and non-enzymatic cell separation solution with the same volume, the concentration of the non-enzymatic cell separation solution is 0.1-0.5%, and the pH value of the saline is 7.4;
s23: gently shaking the mixture at room temperature for 5-10 minutes to release the stem cells from the pellet and uniformly disperse in the buffer;
s24: after gentle shaking, the mixture was allowed to stand for 5 minutes to allow the heavier non-stem cell components to settle to the bottom of the vessel, and then the supernatant liquid, which contained the isolated stem cells, was collected.
Further, the step S3 specifically includes:
s31: transferring the upper liquid collected in the step S24 to a new sterile container, and performing centrifugal treatment in a temperature-controlled centrifugal machine, wherein the temperature of the centrifugal machine is initially set to be 4 ℃;
S33: starting the centrifugation process, gradually increasing the temperature of the centrifuge, gradually increasing the temperature from 4 ℃ to 10 ℃ in the first 5 minutes, and increasing the temperature from 10 ℃ to room temperature in the last 5 minutes;
S34: in the whole centrifugation process, the centrifugation speed is kept constant at 300g, and the total centrifugation time is controlled at 10-15 minutes;
S35: after centrifugation is completed, the supernatant is removed to collect the stem cells that have settled at the bottom of the vessel.
Further, the step S4 specifically includes:
S41: transferring the stem cell pellet collected in S35 to a new sterile container;
s42: adding a separation medium of a polysaccharide solution having a specific gravity of 1.077g/mL to the vessel, and gently shaking the vessel to uniformly disperse the stem cell pellet in the separation medium;
S43: under aseptic condition, placing the container in a centrifuge, and setting the centrifugation speed to be 400-500g and the centrifugation time to be 20-30 minutes;
s45: after centrifugation is complete, the different density layers are removed from the vessel and the layer containing stem cells is collected.
Further, the step S5 specifically includes:
s51: transferring the layer of stem cells collected in S45 into a sterile culture vessel;
S52: adding a pre-prepared culture medium into a culture container, wherein the culture medium comprises 10-20% of fetal bovine serum, and 1-10ng/mL of human fibroblast growth factor and epidermal growth factor;
S53: conditions of the culture environment were adjusted, including setting the temperature to 37 ℃, maintaining the carbon dioxide concentration at 5%, and controlling the humidity at 95%;
S54: the medium was changed periodically, every 48-72 hours.
Further, the applying a low-intensity pulsed electric field technique in S6 includes:
S61: placing the cultured stem cells in an electric field device;
S62: the device is regulated to generate a low-intensity pulsed electric field, wherein the electric field intensity is specifically set to be 1-5V/cm, and the pulse frequency is 1-10 Hz;
s63: the stem cells are exposed to the electric field environment of S62 for 10-30 minutes.
Further, the step S7 specifically includes:
S71: during stem cell culture, adding a cell biomarker which is a fluorescent dye or an antibody into the culture medium every 24-48 hours;
S72: using a fluorescence microscope or a confocal microscope to observe and capture an image of the stem cells;
S73: the captured images are automatically analyzed using image analysis software to quantify the number, size, morphology of stem cells and expression level of markers, and the quality and proliferation status of stem cells are evaluated according to the image analysis result and expression of cell markers.
The invention has the beneficial effects that:
According to the invention, mechanical and chemical damage to stem cells is remarkably reduced by adopting a non-enzymatic cell separation method and a temperature-controlled centrifugation process, so that the integrity and bioactivity of the cells are maintained, and the mild treatment method avoids the cell membrane damage and functional degradation common in the traditional cell separation technology, so that the quality and survival rate of the stem cells are improved, which is important for subsequent experimental research or clinical application, because the high-quality stem cells are more likely to maintain the characteristics in vitro culture and show better therapeutic effects in clinical application.
In the invention, the stem cells are cultivated in the microenvironment of specific nutritional factors and growth factors in the cultivation stage, so that the proliferation and maturation of the stem cells can be effectively promoted, in addition, the biological activity of the stem cells is further enhanced by applying a low-intensity pulsed electric field technology, so that the proliferation capacity and the therapeutic potential of the cells are improved, and the optimized cultivation conditions not only improve the number of the stem cells, but also keep the undifferentiated state of the stem cells, which is particularly important for research and treatment.
The invention allows scientific researchers and clinicians to monitor the quality and quantity of stem cells in real time through the applied cell biomarker and high-resolution microscopic imaging technology, and the real-time monitoring not only provides instant feedback of the stem cell culture process, but also enables optimization of culture conditions to be more accurate and efficient.
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In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a stem cell collection method with little damage to stem cells according to an embodiment of the present invention.
Detailed Description
The present invention will be further described in detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
It is to be noted that unless otherwise defined, technical or scientific terms used herein should be taken in a general sense as understood by one of ordinary skill in the art to which the present invention belongs. The terms "first," "second," and the like, as used herein, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof, but does not exclude other elements or items. The terms "connected" or "connected," and the like, are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", etc. are used merely to indicate relative positional relationships, which may also be changed when the absolute position of the object to be described is changed.
Example 1
As shown in fig. 1, a stem cell collection method with little damage to stem cells comprises the following steps:
S1: collecting a biological sample containing stem cells from bone marrow and physically separating by low-speed centrifugation to reduce damage to cells;
s2: placing the sample obtained in the step S1 into a special buffer solution, and treating the biological sample to separate stem cells;
S3: optimizing the collection efficiency of stem cells by gradually increasing the temperature through a temperature-controlled centrifugation process;
S4: separating stem cells from other cell types by differential sedimentation based on the difference in optical density of the cells;
s5: transferring the stem cells isolated in S4 into a microenvironment containing specific trophic factors and growth factors to promote proliferation and maturation thereof;
s6: applying a low-intensity pulsed electric field technique in a culture environment to enhance the bioactivity and proliferation capacity of stem cells;
s7: and the quality and the number of the stem cells are monitored in real time by adopting a cell biomarker and a high-resolution microscopic imaging technology, so that the stem cells are effectively collected.
The physical separation by low-speed centrifugation in S1 specifically includes:
S11: transferring the collected bone marrow sample into a sterile container, extracting the sample to 8 ml in volume, and adding an equal volume of anticoagulant to prevent blood coagulation;
s12: placing the container containing the sample in a centrifuge under aseptic conditions, and setting the centrifugation speed to be 230g and the centrifugation time to be 13 minutes to realize physical separation;
s13: after centrifugation is completed, the upper plasma and middle leukocytes are removed, leaving a bottom pellet containing stem cells.
The step S2 specifically comprises the following steps:
S21: transferring the bottom sediment containing the stem cells obtained in the step S1 to a new sterile container;
S22: adding a special buffer solution into a sterile container, wherein the buffer solution consists of phosphate buffer physiological saline and a non-enzymatic cell separation solution with the concentration of 0.3%, and the pH value of the saline is 7.4, so as to maintain the integrity of stem cells and reduce biochemical damage;
S23: gently shaking the mixture at room temperature for 7 minutes to release the stem cells from the pellet and uniformly disperse in the buffer;
S24: after gentle shaking, the mixture was allowed to stand for 5 minutes to allow the heavier non-stem cell components to settle to the bottom of the vessel, and then the supernatant liquid, which contained the isolated stem cells, was collected and ready for further processing.
The step S3 specifically comprises the following steps:
s31: transferring the upper liquid collected in the step S24 to a new sterile container, and performing centrifugal treatment in a temperature-controlled centrifugal machine, wherein the temperature of the centrifugal machine is initially set to be 4 ℃;
S33: starting the centrifugation process, gradually increasing the temperature of the centrifuge, gradually increasing the temperature from 4 ℃ to 10 ℃ in the first 5 minutes, and increasing the temperature from 10 ℃ to room temperature in the last 5 minutes;
S34: in the whole centrifugation process, the centrifugation speed is kept constant at 300g, and the total centrifugation time is controlled at 13 minutes;
S35: after centrifugation is completed, the supernatant is removed to collect the stem cells that have settled at the bottom of the vessel.
The step S4 specifically comprises the following steps:
S41: transferring the stem cell pellet collected in S35 to a new sterile container;
s42: adding a separation medium of a polysaccharide solution having a specific gravity of 1.077g/mL to the vessel, and gently shaking the vessel to uniformly disperse the stem cell pellet in the separation medium;
s43: placing the container in a centrifuge under aseptic conditions and setting a centrifugation speed of 450g for 25 minutes to effect density-based cell separation;
s45: after centrifugation is complete, the different density layers are removed from the vessel and the layer containing stem cells is collected.
The step S5 specifically comprises the following steps:
s51: transferring the layer of stem cells collected in S45 into a sterile culture vessel;
S52: adding a pre-formulated medium comprising 15% Fetal Bovine Serum (FBS), and 5ng/mL human fibroblast growth factor (hFGF) and Epidermal Growth Factor (EGF) to a culture vessel;
S53: conditions of the culture environment were adjusted, including setting the temperature to 37 ℃, maintaining the carbon dioxide concentration at 5%, and controlling the humidity at 95%;
s54: the medium was changed periodically, every 60 hours, to maintain proper nutrient and growth conditions.
The applying a low-intensity pulsed electric field technique in S6 includes:
S61: placing the cultured stem cells in an electric field device;
s62: the device is regulated to generate a low-intensity pulse electric field, wherein the electric field intensity is specifically set to be 3V/cm, and the pulse frequency is 5Hz;
s63: the stem cells are exposed to the electric field environment of S62 for 20 minutes, once per culture cycle, and after the electric field treatment, the stem cells are returned to the original culture environment and the conventional culture conditions are continued.
The step S7 specifically comprises the following steps:
S71: during stem cell culture, a cell biomarker, which is a fluorescent dye for labeling cell activity, is added to the culture medium every 36 hours;
S72: using a fluorescence microscope, observing and capturing an image of the stem cells;
S73: the captured images are automatically analyzed using image analysis software to quantify the number, size, morphology of stem cells and expression level of markers, and the quality and proliferation status of stem cells are evaluated according to the image analysis result and expression of cell markers.
Example 2
Step 1: collecting biological sample containing stem cells from umbilical cord blood, wherein the volume is 5 ml, adding an equal volume of anticoagulant to prevent blood coagulation, transferring the sample into a sterile container, centrifuging at 200g speed in a centrifuge for 10 minutes, removing upper plasma and middle white blood cells, and retaining bottom sediment containing stem cells;
Step 2: transferring the bottom sediment obtained in the step 1 into a new sterile container, adding a buffer solution consisting of an equal volume of phosphate buffer physiological saline and 0.1% concentration non-enzymatic cell separation solution, adjusting the pH value to 7.4, slightly shaking the mixture at room temperature for 5 minutes, releasing stem cells from the sediment and uniformly dispersing the stem cells in the buffer solution, standing for 5 minutes, settling the non-stem cells, and collecting the liquid containing the stem cells at the upper layer;
Step 3: transferring the upper liquid collected in the step 2 to a new sterile container, centrifuging in a temperature-controlled centrifuge, setting the initial temperature to 4 ℃, gradually increasing the temperature to 10 ℃ in the first 5 minutes, increasing the temperature to room temperature in the last 5 minutes, keeping the centrifuging speed at 300g for 10 minutes, removing the upper liquid after centrifuging, and collecting the precipitated stem cells;
Step 4: transferring the stem cell sediment collected in the step 3 into a new sterile container, adding a glycan solution with the specific gravity of 1.077g/mL as a separating medium, gently shaking the container to uniformly disperse the stem cell sediment, centrifuging under the sterile condition, setting the speed to 400g for 20 minutes, removing layers with different densities after centrifuging, and collecting a layer containing the stem cells;
Step 5: transferring the stem cell layer collected in the step 4 into a sterile culture container, adding a pre-prepared culture medium comprising 10% fetal bovine serum, 1ng/mL human fibroblast growth factor and epidermal growth factor into the culture container, and regulating the conditions of a culture environment: the temperature is 37 ℃, the carbon dioxide concentration is 5%, the humidity is 95%, and the culture medium is replaced periodically every 48 hours;
Step 6: the cultured stem cells are placed in an electric field device, the regulating device generates an electric field strength of 1V/cm and a pulse frequency of 1Hz, the stem cells are exposed in the electric field environment for 10 minutes to enhance the bioactivity and proliferation capacity,
Step 7: during the culturing, antibodies were added to the medium every 24 hours as cell biomarkers, then images of stem cells were observed and captured using a confocal microscope, and the captured images were automatically analyzed using image analysis software to quantify the number, size, morphology and marker expression level of stem cells, and the quality and proliferation status of stem cells were evaluated based on the results of the image analysis and the cell marker expression.
Example 3
Step 1: collecting biological sample containing stem cells from bone marrow, wherein the volume is 10 ml, adding an equal volume of anticoagulant to prevent blood coagulation, transferring the sample into a sterile container, centrifuging in a centrifuge at 250g for 15min, removing upper plasma and middle white blood cells, and retaining bottom sediment containing stem cells;
Step 2: transferring the bottom sediment obtained in the step 1 into a new sterile container, adding a buffer solution consisting of an equal volume of phosphate buffer physiological saline and 0.5% concentration non-enzymatic cell separation solution, adjusting the pH value to 7.4, slightly shaking the mixture at room temperature for 10 minutes, releasing stem cells from the sediment and uniformly dispersing the stem cells in the buffer solution, standing for 5 minutes, settling the non-stem cells, and collecting the liquid containing the stem cells at the upper layer;
Step 3: transferring the upper liquid collected in the step 2 to a new sterile container, centrifuging in a temperature-controlled centrifuge, setting the initial temperature to 4 ℃, gradually increasing the temperature to 10 ℃ in the first 5 minutes, increasing the temperature to room temperature in the last 5 minutes, keeping the centrifuging speed at 300g for 15 minutes, removing the upper liquid after centrifuging, and collecting the precipitated stem cells;
step 4: transferring the stem cell sediment collected in the step 3 into a new sterile container, adding a glycan solution with the specific gravity of 1.077g/mL as a separating medium, gently shaking the container to uniformly disperse the stem cell sediment, centrifuging under the sterile condition, setting the speed to be 500g, setting the time to be 30 minutes, removing layers with different densities after centrifuging, and collecting a layer containing the stem cells;
Step 5: transferring the stem cell layer collected in the step 4 into a sterile culture container, adding a pre-prepared culture medium comprising 20% fetal bovine serum, 10ng/mL human fibroblast growth factor and epidermal growth factor into the culture container, and regulating the conditions of a culture environment: the temperature is 37 ℃, the carbon dioxide concentration is 5%, the humidity is 95%, and the culture medium is replaced periodically every 72 hours;
Step 6: the cultured stem cells are placed in an electric field device, the regulating device generates an electric field strength of 5V/cm and a pulse frequency of 10Hz, the stem cells are exposed in the electric field environment for 30 minutes to enhance the bioactivity and proliferation capacity,
Step 7: during the culturing, fluorescent dye is added to the culture medium every 48 hours as a cell biomarker, then an image of stem cells is observed and captured using a fluorescent microscope, and the captured image is automatically analyzed using image analysis software to quantify the number, size, morphology and marker expression level of the stem cells, and the quality and proliferation state of the stem cells are evaluated according to the image analysis result and the cell marker expression.
Table 1 comparison of final experimental data
| Experimental items | Example 1 | Example 2 | Example 3 |
| Stem cell collection efficiency (%) | 92 | 87 | 90 |
| Stem cell Activity score | 9.5 (Full 10 minutes) | 8.7 (Full 10 minutes) | 9.2 (Full 10 minutes) |
| Stem cell proliferation rate | 1.8 Times/24 hours | 1.5 Times/24 hours | 1.7 Times/24 hours |
| Stem cell maturation time (Tian) | 15 | 18 | 16 |
| Stem cell morphology consistency | 95％ | 90％ | 93％ |
From table 1 above, it can be seen that example 1 shows the optimal stem cell collection efficiency (92%), indicating that the method is more efficient in capturing and retaining cells, and in addition, the stem cell activity score is highest (9.5 minutes), indicating that the damage to the cell activity during the collection is the smallest, and higher biological activity is maintained, and the stem cells of example 1 show a proliferation rate 1.8 times faster than other examples in terms of the proliferation rate of the stem cells, meaning that more stem cells can be obtained in the same time, the stem cell maturation time is 15 days, shorter than examples 2 and 3, showing a more efficient maturation process, and the stem cell morphology consistency is 95%, indicating that the stem cells obtained by the method are more uniform in morphology, which is beneficial for subsequent applications and researches.
In summary, example 1 not only shows excellent collection efficiency and activity of stem cells, but also shows better performance in terms of proliferation rate, maturation time and morphological consistency, and is the best choice among the three examples.
The present invention is intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.
Claims (8)
1. The stem cell collection method with small damage to the stem cells is characterized by comprising the following steps:
S1: collecting a biological sample containing stem cells from bone marrow or umbilical cord blood, and performing physical separation by low-speed centrifugation;
s2: placing the sample obtained in the step S1 into a special buffer solution, and treating the biological sample to separate stem cells;
S3: optimizing the collection efficiency of stem cells by gradually increasing the temperature through a temperature-controlled centrifugation process;
s4: separating stem cells from other cell types by adopting a differential sedimentation method;
s5: transferring the stem cells isolated in S4 into a microenvironment containing specific trophic factors and growth factors to promote proliferation and maturation thereof;
s6: applying a low-intensity pulsed electric field technique in a culture environment to enhance the bioactivity and proliferation capacity of stem cells;
s7: and the quality and the number of the stem cells are monitored in real time by adopting a cell biomarker and a high-resolution microscopic imaging technology, so that the stem cells are effectively collected.
2. The method for collecting stem cells with little damage to stem cells according to claim 1, wherein the physical separation by low-speed centrifugation in S1 specifically comprises:
S11: transferring the collected bone marrow sample or umbilical cord blood sample into a sterile container, extracting the sample with a volume of 5-10 ml, and adding an equal volume of anticoagulant to prevent blood coagulation;
S12: placing the container containing the sample in a centrifuge under aseptic condition, and setting the centrifugation speed to be 200-250g and the centrifugation time to be 10-15 minutes;
s13: after centrifugation is completed, the upper plasma and middle leukocytes are removed, leaving a bottom pellet containing stem cells.
3. The method for collecting stem cells with little damage to stem cells according to claim 2, wherein S2 specifically comprises:
S21: transferring the bottom sediment containing the stem cells obtained in the step S1 to a new sterile container;
S22: adding a special buffer solution into a sterile container, wherein the buffer solution consists of phosphate buffer physiological saline and non-enzymatic cell separation solution with the same volume, the concentration of the non-enzymatic cell separation solution is 0.1-0.5%, and the pH value of the saline is 7.4;
s23: gently shaking the mixture at room temperature for 5-10 minutes to release the stem cells from the pellet and uniformly disperse in the buffer;
s24: after gentle shaking, the mixture was allowed to stand for 5 minutes to allow the heavier non-stem cell components to settle to the bottom of the vessel, and then the supernatant liquid, which contained the isolated stem cells, was collected.
4. The method for collecting stem cells with little damage to stem cells according to claim 3, wherein the step S3 specifically comprises:
s31: transferring the upper liquid collected in the step S24 to a new sterile container, and performing centrifugal treatment in a temperature-controlled centrifugal machine, wherein the temperature of the centrifugal machine is initially set to be 4 ℃;
S33: starting the centrifugation process, gradually increasing the temperature of the centrifuge, gradually increasing the temperature from 4 ℃ to 10 ℃ in the first 5 minutes, and increasing the temperature from 10 ℃ to room temperature in the last 5 minutes;
S34: in the whole centrifugation process, the centrifugation speed is kept constant at 300g, and the total centrifugation time is controlled at 10-15 minutes;
S35: after centrifugation is completed, the supernatant is removed to collect the stem cells that have settled at the bottom of the vessel.
5. The method for stem cell collection with little damage to stem cells according to claim 4, wherein S4 specifically comprises:
S41: transferring the stem cell pellet collected in S35 to a new sterile container;
s42: adding a separation medium of a polysaccharide solution having a specific gravity of 1.077g/mL to the vessel, and gently shaking the vessel to uniformly disperse the stem cell pellet in the separation medium;
S43: under aseptic condition, placing the container in a centrifuge, and setting the centrifugation speed to be 400-500g and the centrifugation time to be 20-30 minutes;
s45: after centrifugation is complete, the different density layers are removed from the vessel and the layer containing stem cells is collected.
6. The method for stem cell collection with little damage to stem cells according to claim 5, wherein S5 specifically comprises:
s51: transferring the layer of stem cells collected in S45 into a sterile culture vessel;
S52: adding a pre-prepared culture medium into a culture container, wherein the culture medium comprises 10-20% of fetal bovine serum, and 1-10ng/mL of human fibroblast growth factor and epidermal growth factor;
S53: conditions of the culture environment were adjusted, including setting the temperature to 37 ℃, maintaining the carbon dioxide concentration at 5%, and controlling the humidity at 95%;
S54: the medium was changed periodically, every 48-72 hours.
7. The method of claim 6, wherein applying a low-intensity pulsed electric field technique in S6 comprises:
S61: placing the cultured stem cells in an electric field device;
S62: the device is regulated to generate a low-intensity pulsed electric field, wherein the electric field intensity is specifically set to be 1-5V/cm, and the pulse frequency is 1-10 Hz;
s63: the stem cells are exposed to the electric field environment of S62 for 10-30 minutes.
8. The method for stem cell collection with little damage to stem cells of claim 7, wherein S7 specifically comprises:
S71: during stem cell culture, adding a cell biomarker which is a fluorescent dye or an antibody into the culture medium every 24-48 hours;
S72: using a fluorescence microscope or a confocal microscope to observe and capture an image of the stem cells;
S73: the captured images are automatically analyzed using image analysis software to quantify the number, size, morphology of stem cells and expression level of markers, and the quality and proliferation status of stem cells are evaluated according to the image analysis result and expression of cell markers.
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