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Method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue

An adipose tissue and stem cell technology, applied in the field of biomedicine, can solve the problems of inability to ensure biological safety and quality control, inability to effectively retain adult stem cells/progenitor cells, and no product quality requirements, achieving a simple and reliable method and avoiding cross-contamination. , the effect of operating norms

Active Publication Date: 2010-04-14
NINGXIA ZHONGLIANDA BIOPHYSICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, although there are already methods for extracting mesenchymal stem cells from placental umbilical cord tissue and methods for obtaining trophic layer stem cells from placental umbilical cord tissue, they are all laboratory techniques, and there is still a long way to go before the industrialization of products; Second, these methods can only obtain a single type of stem cells, and cannot effectively retain all adult stem cells / progenitor cells; third, because the laboratory technology has no operating standards and product quality requirements, it cannot guarantee biological safety in clinical application and quality control

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Kit composition:

[0012] 1. The precipitant is an aqueous solution of 5% hydroxyethyl starch and 0.4% calcium gluconate, the pH value is 7.4±0.2, and the osmotic pressure is 279mOsm / kg.

[0013] 2. Sodium diatrizoate-polysucrose 400 # : 5% Ficoll 400 # etc., the mixed solution of 12.5% ​​sodium diatrizoate has a density of 1.077-1.080 and a pH value of 7.4±0.2.

[0014] 3. Cell maintenance solution Phosphate buffer solution, pH value is 7.4±0.2.

[0015] 4. Collagenase II or III or IV 50-100mg or 0.25% trypsin 10ml.

[0016] 5. The kit is also equipped with a one-time sterilized tissue pulverizing bucket, pulverizer cutter head, Chua filter, 50ml siliconized centrifuge tube, 10ml and 25ml pipettes, and a combined package consisting of cell transfer tubes. Single-use packaging sealed in a blister box. Use gamma rays to sterilize or ethylene oxide to sterilize. Each kit comes with a set of disposable supplies.

[0017] The above 1-3 reagents were prepared and ster...

Embodiment 2

[0024] Kit composition:

[0025] 1. The precipitant is an aqueous solution of 7% hydroxyethyl starch and 0.2% calcium gluconate, the pH value is 7.4±0.2, and the osmotic pressure is 279mOsm / kg.

[0026] 2. Sodium diatrizoate-polysucrose 400 # : 6% Ficoll 400 # etc., 10% sodium diatrizoate mixture, density 1.077-1.080, pH value 7.4±0.2, osmotic pressure 279mOsm / kg.

[0027] 3. Cell maintenance solution, phosphate buffer.

[0028] 4. Collagenase II or III or IV 50-100mg or 0.25% trypsin 10ml. .

[0029] 5. The kit is also equipped with a one-time sterilized tissue crushing bucket, crushing head, Chua filter, 50ml siliconized centrifuge tube, 10ml and 25ml pipettes, and a combined package consisting of cell transfer tubes. Single-use packaging sealed in a blister box. Use gamma rays to sterilize or ethylene oxide to sterilize. Each kit comes with a set of disposable supplies.

[0030] After the above 1-3 reagents are prepared, they are sterilized by autoclaving at 121°C f...

Embodiment 3

[0036] Kit composition:

[0037] 1. The precipitant is an aqueous solution of 5% hydroxyethyl starch and 0.4% calcium gluconate, the pH value is 7.4±0.2, and the osmotic pressure is 279mOsm / kg.

[0038] 2. Sodium diatrizoate-polysucrose 400 # : 5% Ficoll 400 # etc., 12.5% ​​sodium diatrizoate mixture, density 1.077-1.080, pH value 7.4±0.2, osmotic pressure 279mOsm / kg.

[0039] 3. Phosphate buffer solution for cell maintenance.

[0040] 4. Collagenase II or III or IV 50-100mg or 0.25% trypsin 10ml.

[0041] 5. The kit is also equipped with a one-time sterilized tissue crushing bucket, crushing head, Chua filter, 50ml siliconized centrifuge tube, 10ml and 25ml pipettes, and a combined package consisting of cell transfer tubes. Single-use packaging sealed in a blister box. Use gamma rays to sterilize or ethylene oxide to sterilize. Each kit comes with a set of disposable supplies.

[0042] The above 1-3 reagents were prepared and sterilized by autoclaving at 121°C for 35 m...

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Abstract

The invention relates to a method for separating and extracting stem cells from a placenta, umbilical cord or adipose tissue, which comprises the following process steps: firstly mixing the placenta, umbilical cord or adipose tissue and a cell maintenance fluid according to the proportion by weight of 2.5-4:1, putting the mixture into a tissue crushing barrel, adding collagenase after crushing, uniformly mixing, hatching at the temperature of 37 DEG C, filtering, adding a precipitator, sucking a supernatant fluid after settling, centrifuging, removing the supernatant fluid, adding concentrated cells into a liquid of diatrizoate sodium-ficoll 400#, then centrifuging, collecting 10-15 ml of intermediate cell layer, washing with the cell maintenance fluid, counting the collected cells, and providing the cells for clinical use when the cell survival ratio is more than or equal to 95 percent. The invention not only realizes the separation and the extraction of all stem cells from the placenta, umbilical cord or adipose tissue, but also realizes industrialized production, and enables doctors to conveniently, safely and canonically obtain the adult stem cells in clinic and use the adult stem cells for treating the diseases of patients as using medicaments, thereby solving the bottleneck problem of difficult obtainment of adult stem cells in clinic and popularizing a cell treatment technology.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue. Background technique [0002] At present, there are many methods for isolating cells from human bone marrow and umbilical cord blood in the world. The applicant has also applied for two patented technologies, such as "Bone Marrow Cord Blood Stem Cell In Vitro Separation Kit and Its Application Method" (application number 200710137781.9) and "One A Nucleated Cell In Vitro Isolation Kit and Its Application Method" (Application No. 200610106875.5). These technologies provide technical support for obtaining stem cells from human bone marrow and fetal umbilical cord blood, and open up broad prospects for the clinical application of stem cells. [0003] However, studies in recent years have found that human placenta and umbilical cord not only contain a large number of stem cells in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/0775
Inventor 王沁怡王怀林
Owner NINGXIA ZHONGLIANDA BIOPHYSICS
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