CN113583950A - Method for preparing stem cell active factor and application thereof - Google Patents
Method for preparing stem cell active factor and application thereof Download PDFInfo
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- 210000000130 stem cell Anatomy 0.000 title claims abstract description 203
- 238000000034 method Methods 0.000 title claims abstract description 119
- 238000004140 cleaning Methods 0.000 claims abstract description 82
- 102000029816 Collagenase Human genes 0.000 claims abstract description 35
- 108060005980 Collagenase Proteins 0.000 claims abstract description 35
- 229960002424 collagenase Drugs 0.000 claims abstract description 35
- 239000001963 growth medium Substances 0.000 claims abstract description 34
- 239000003223 protective agent Substances 0.000 claims abstract description 34
- 230000003321 amplification Effects 0.000 claims abstract description 23
- 230000029087 digestion Effects 0.000 claims abstract description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 23
- 239000013049 sediment Substances 0.000 claims abstract description 23
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 23
- 210000003954 umbilical cord Anatomy 0.000 claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 22
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 238000003306 harvesting Methods 0.000 claims abstract description 12
- 239000000843 powder Substances 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 45
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 33
- 238000005119 centrifugation Methods 0.000 claims description 33
- 239000008055 phosphate buffer solution Substances 0.000 claims description 33
- 238000005406 washing Methods 0.000 claims description 23
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 21
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 21
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 21
- 235000017281 sodium acetate Nutrition 0.000 claims description 21
- 239000001632 sodium acetate Substances 0.000 claims description 21
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 11
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 11
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 238000010008 shearing Methods 0.000 claims description 11
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- 210000004027 cell Anatomy 0.000 abstract description 9
- 230000000052 comparative effect Effects 0.000 description 17
- 239000011324 bead Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 210000003014 totipotent stem cell Anatomy 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000002444 unipotent stem cell Anatomy 0.000 description 1
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention discloses a method for preparing stem cell active factors and application thereof, belonging to the technical field of cell engineering. It comprises the following steps: (1) primary acquisition of stem cells: taking umbilical cord of parturient, adding collagenase, and performing digestion treatment on an oscillator; (2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage, taking the sediment of the lower layer, and cleaning; (3) three-stage harvesting of stem cells: transferring the secondary obtained stem cells into a culture medium, and performing amplification culture after culture; (4) extraction of active factors: transferring the stem cells obtained in the third stage into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering and sterilizing, and then crushing by using an ultrasonic crusher and performing ultrafiltration treatment; (5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder. The prepared stem cell active factor meets the use requirement.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to a method for preparing stem cell active factors and application thereof.
Background
Cells (stem cells) are a type of pluripotent cells with self-replicating capacity (self-rejuvenating). Under certain conditions, it can differentiate into a variety of functional cells. The stem cells are classified into embryonic stem cells (ES cells) and adult stem cells (somatics cells) according to the developmental stage in which the stem cells are located. The stem cells are classified into three categories according to their developmental potential: totipotent Stem Cells (TSC), pluripotent stem cells (pluripotent stem cells) and unipotent stem cells (multipotent stem cells). Stem cells (Stem cells) are insufficiently differentiated and immature cells, have the potential function of regenerating various tissues, organs and human bodies, and are called "universal cells" in the medical field.
The conventional stem cell factor sorting technology is that magnetic bead sorting is common, but in the conventional sorting, magnetic beads are cultured together with stem cells, namely the magnetic beads are not separated from the stem cells, so that certain influence is certainly generated on the later production of the stem cells, and although some methods are adopted, such as adding some bacteria or other substances for degradation, other exogenous proteins and other components are equivalently added, so that secondary pollution to the stem cells is caused, and the problems are not actually solved.
Disclosure of Invention
Problems to be solved
Aiming at the problems in the prior art, the invention provides a method for preparing stem cell active factors and application thereof, the process is simple, and the prepared stem cell active factors meet the use requirements.
Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
A method for preparing stem cell active factor, comprising the steps of:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 5: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
has the beneficial effects of
Compared with the prior art, the invention has the beneficial effects that:
the stem cell active factor obtained by the extraction process is mainly used in laboratories or small factories, and the purity can meet the requirements. After comparing with traditional technique, can find, make full use of the mode that tertiary was obtained, rationally set up effectual collagenase, set up the mixed washing of three kinds of substances of second normal saline, sodium acetate and polyvinyl alcohol, satisfy the user demand of stem cell active factor.
Detailed Description
The invention is further described with reference to specific examples.
Example 1
Best mode for carrying out the invention
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 5: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
comparative example 1
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 5: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 100: 1.
comparative example 2
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 5: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 200: 1.
comparative example 3
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 80: 5: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
comparative example 4
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 15: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
comparative example 5
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 5: 40.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
comparative example 6
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of physiological saline and polyvinyl alcohol;
wherein the mass ratio of the physiological saline to the polyvinyl alcohol is 70: 35.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
comparative example 7
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline and sodium acetate;
wherein the mass ratio of the normal saline to the sodium acetate is 70: 5.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
comparative example 8
The method for preparing stem cell active factors of the embodiment comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
In the above-mentioned method for preparing stem cell active factor and its application,
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
In the above-mentioned method for preparing stem cell active factor and its application,
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the culture medium in the step (3) is a DMEM culture medium.
In the above-mentioned method for preparing stem cell active factor and its application,
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
in the above-mentioned method for preparing stem cell active factor and its application,
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
In the above-mentioned method for preparing stem cell active factor and its application,
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
In the above-mentioned method for preparing stem cell active factor and its application,
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 20: 20.
in the above-mentioned method for preparing stem cell active factor and its application,
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
example 2
The product prepared in example 1 and the products prepared in comparative examples 1 to 8 were selected and subjected to the following tests:
referring to the Chinese invention patent, the application number: CN201810318819.0, publication No.: CN110354057A discloses a lyophilized powder of stem cell active factor.
Wherein the extraction rate of the active factors in example 1 is 5.2%;
wherein the extraction rate of the active factor in comparative example 1 is 4.9%;
wherein the extraction rate of the active factor in comparative example 2 is 4.8%;
wherein the extraction rate of the active factor in comparative example 3 is 4.3%;
wherein the extraction rate of the active factor in comparative example 4 is 4.0%;
wherein the extraction rate of the active factor in comparative example 5 is 3.2%;
wherein the extraction rate of the active factor in comparative example 6 is 2.5%;
wherein the extraction rate of the active factor in comparative example 7 is 2.1%;
wherein the extraction rate of the active factor in comparative example 8 is 1.9%.
While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.
Claims (10)
1. A method for preparing stem cell active factors, which is characterized by comprising the following steps:
the method comprises the following steps:
(1) primary acquisition of stem cells: taking umbilical cord of a parturient, cleaning and shearing, then adding collagenase, and carrying out digestion treatment on the umbilical cord in an oscillator;
(2) secondary acquisition of stem cells: centrifuging the stem cells obtained in the first stage in the step (1), taking the sediment of the lower layer, and washing the sediment by using a PBS (phosphate buffer solution);
(3) three-stage harvesting of stem cells: transferring the stem cells obtained in the second stage in the step (2) into a culture medium, and performing amplification culture after culture;
(4) extraction of active factors: transferring the stem cells obtained in the third stage in the step (3) into a cleaning tank, performing centrifugal treatment, collecting supernate, filtering for sterilization, then crushing by using an ultrasonic crusher, and performing ultrafiltration treatment;
(5) preservation treatment: and (4) cleaning the active factors extracted in the step (4) by using a cleaning solution, adding a protective agent after cleaning, and placing in a freeze dryer to obtain freeze-dried powder.
2. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the solution used for cleaning in the step (1) is Hank's balanced salt solution;
the temperature of the solution used for washing in step (1) was 4 ℃.
3. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the adding mass of the collagenase in the step (1) is 0.05 percent;
the temperature of the oscillator in the step (1) is 37 ℃;
the digestion time in the step (1) is 18 min;
collagenase purchased from Beijing Sorleibao science and technology Limited is collagenase I, with a product number of C8140.
4. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the rotating speed of the centrifugation in the step (2) is 15000 rpm;
the temperature of centrifugation in the step (2) is 4 ℃;
the temperature of the PBS buffer in step (2) was 4 ℃.
5. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the culture medium in the step (3) is a DMEM culture medium.
6. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the conditions for the amplification culture in the step (3) are as follows:
37℃、5%CO2、7d。
7. the method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the centrifugal force of the centrifugal treatment in the step (4) is 15000 g;
the temperature of the centrifugation in the step (4) was 4 ℃.
8. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the aperture of the ultrafiltration treatment in the step (4) is 200 daltons.
9. The method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the cleaning solution in the step (5) comprises a mixture of normal saline, sodium acetate and polyvinyl alcohol;
wherein the mass ratio of the normal saline to the sodium acetate to the polyvinyl alcohol is 70: 5: 35.
10. the method for preparing stem cell active factor and the use thereof according to claim 1, wherein:
the protective agent in the step (5) is glycerol, wherein the mass ratio of the protective agent to the active factors is 150: 1.
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Cited By (1)
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| CN117618468A (en) * | 2023-11-22 | 2024-03-01 | 安徽科门生物科技有限公司 | Process and application of alopecia treatment preparation rich in stem cell active factors |
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