CN1597937A - Extracting material of placental villi cell and its application for inducing differentiating of matrax stem cell - Google Patents
Extracting material of placental villi cell and its application for inducing differentiating of matrax stem cell Download PDFInfo
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- CN1597937A CN1597937A CN 200410040280 CN200410040280A CN1597937A CN 1597937 A CN1597937 A CN 1597937A CN 200410040280 CN200410040280 CN 200410040280 CN 200410040280 A CN200410040280 A CN 200410040280A CN 1597937 A CN1597937 A CN 1597937A
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Abstract
The invention relates to a cell extract of human or animal placental amnion and its application to in vitro induced differentiation of mesenchymal stem cells into skin cells, providing a new inducing reagent for stem cell induction technique, obtained by the flow: placental amnion tissue->washing and cutting into pieces->cell homogenizing->cell breaking->depositing->taking supernatant-> separating and eliminating substances with molecular weight greater than above 200 thousand Da split charging. The application of the invention can be an application of organically combining cell extract of placental amnion with existing technique and also that of preparing the cell extract of placental amnion into reagent box, and it shows good induced differentiation ratio at above 90% in the application of the induced differentiation of stem cells into skin cells. The obtained skin cells can cure burn, scald, and other skin destructive diseases.
Description
Technical field
The present invention relates to biomass cells extract and utilisation technology, be specifically related to human or animal's placenta amnion cell extract and be divided into utilisation technology in the skin cells in cell induction at mesenchyme.
Background technology
Dermatoplasty regeneration is the effective means of skin injury disease such as the treatment burn, scald of clinical eager demand.Stem cell has the cell of self and multidirectional differentiation potential as a class, has become the new resources that people seek skin cells gradually.
Since 1997, stem-cell research makes important progress, discover that human body mescenchymal stem cell (MSC) can be induced to differentiate into the mature cell of nerve, muscle, skin, cartilage, fat, tendon, insulin secretion, liver, blood and types of organization, be transplanted in the body after amplification is induced and respective organization cell good integration, bring into play specific biological function, can be used for the multiple disease of transplantation treatment.Stem cell is induced the optimal selection that the skin cells after the differentiation is transplanted will become clinical skin injury disease therapeutic, can be used for skin injury diseases such as transplantation treatment burn, scald.
Plasticity-about the derived from bone marrow stem cell is that differentiation potential has obtained following evidence: (1) in cerebral tissue, discovery can be converted into neurocyte with Bone Marrow Stem Cells Transplantation, repairs brain tissue impairment; (2) bone marrow stem cell is at the external insulin-like cell that is induced to differentiate into excreting insulin; (3) marrow hemopoietic stem cells of purifying is transplanted in the embryonic tissue, can enter in the various tissues with fetal development, is divided into the mature cell of respective organization type; (4) the insulin secretion source of human stem cell is in hemopoietic stem cell; (5) stem cell is placed on the membrane film cultivate, can be induced to differentiate into skin cells; (6) MSC is transplanted to the burned skin surface of a wound, can promotes injury repairing.
The stem cell of above-mentioned result of study prompting derived from bone marrow has the potential that is converted into skin cells.
Existing directional induction mescenchymal stem cell is divided into the report of skin cells, and method is that external mescenchymal stem cell is inoculated in the placenta amnion tissue, after cultivating, finds that mescenchymal stem cell is induced to differentiate into skin cells.Though it is clinical that such laboratory study achievement can not directly apply to, its prompting, the placenta amnion tissue can provide microenvironment for mescenchymal stem cell is induced to differentiate into skin cells.
Relevant noun note:
(1) stem cell: be the cells of origin of various mature cells, according to the source and the difference of differentiation potential, be divided into embryonic stem cell (embryonic stemcells, ES) and adult stem cell (dult stem cells, ASCs) two classes.The potential amplification is arranged and induce the differentiation performance.
(2) mescenchymal stem cell (mesenchymal stem cells, MSCs is abbreviated as MSC): be to study more ASCs at present, extensively be present in the multiple tissue of adult, and have multidirectional differentiation potential, can be induced to differentiate into the mature cell of broad variety, difference in functionality in vivo and in vitro.Can reach more than 50 times multiplication and keep the potential of its multidirectional differentiation external.
(3) DMEM and F12 substratum: cell cultures general basic substratum.
(4) ovum extract: extract from human or animal's ovum, the complex mixture that the molecular weight size does not normally wait contains multiple promotion, regulation and control and inducing cell growth and differentiation factor.Can be used as nutritive medium uses.It is in 200410033182.9 the patent application that the technical scheme of this technology is documented in number of patent application.
(5) stem cell is induced differentiation: behind co-cultivation certain hour under specified temp, humidity and the carbon dioxide conditions, differentiation of stem cells is a mature cell with natural compounds or cytokine etc. and stem cell.Different inducible factors induce back gained mature cell type different with condition.
Summary of the invention
The invention provides a kind of placenta amnion tissue of the human or animal body that has drawn from, through artificial that extract, make the placenta amnion cell extract of molecular weight less than 200,000 dalton (Da); The present invention also provides this placenta amnion cell extract to be induced to differentiate in the skin cells utilisation technology as inductor at external MSC, placenta amnion cell extract of the present invention is induced to differentiate in the application of skin cells at MSC, demonstrate the good differentiation rate of inducing, and effective, repeatability and good stability.
The invention technical scheme is as follows:
Human or animal's placenta amnion cell extract mainly comprises following method extraction:
Get human or animal's placenta amnion tissue, clean, shred → cell homogenates → cytoclasis → precipitation or filtration with physiological saline, get the material → determination of protein concentration of supernatant liquor → separation removal greater than the above molecular weight of 200,000 dalton (Da), determination of activity, degerming, frozen or be prepared into the reagent packing.
Concrete grammar is:
(1) gets human or animal's placenta amnion tissue, clean, shred standby with physiological saline;
(2) below 4 ℃, homogenate 3-5 time;
(3) the capable cytoclasis of homogenate is broken fully to cell;
(4) under 1-4 ℃ of condition, place to go sediment and albumen floss are drawn supernatant liquor;
(5) supernatant liquor of Huo Deing is held back and separate to be removed molecular weight greater than the above macromolecular substance of 200,000 dalton (Da), and the molecular weight that makes albumen in the filtered liquid of acquisition and polypeptides matter is less than 200,000 dalton (Da);
(6) the filtered liquid determining the protein quantity of Huo Deing, determination of activity, degerming, frozen or aseptic subpackaged, this is a placenta amnion cell extract of the present invention.
Carry out homogenate after the placenta amnion tissue of step (1) shreds, also can add homogenate again after the dilution of 1-4 times of physiological saline; Step (3) cytoclasis can be used the method for chemistry or physics, as ultrasonic disruption etc., preferred freeze thawing, be to place refrigerator frozen to freezing fully, thoroughly melt not being higher than under 42 ℃ the condition then, preferably water proof is heated, and as above the condition multigelation is 3-5 time, smear microscopic examination routinely proves that cell is broken fully; The separation method of holding back of step (5) can be methods such as high speed centrifugation, electrophoresis, filtering membrane filtration, preferred filtering membrane filters under malleation or condition of negative pressure to be held back, its objective is macromolecular substance is separated, keep the composition of molecular weight less than 200,000 dalton (Da).The requirement of selecting for use of filtering membrane can be held back down the macromolecular substance of molecular weight greater than 200,000 dalton (Da).Placenta amnion cell extract filtrate of the present invention is used the ordinary method generate a reagent, but pulvis or aqua.When making aqua, protein concn greater than 4 mg/ml for well, if protein concentration is low excessively in the aqua, when using in order to satisfy protein concentration, corresponding water capacity increases, and causes other interior composition consumption of nutrient solution of certain volume to reduce, and can not satisfy the nutrient solution ratio requirement.No matter be pulvis or aqua, during application, to the amount of asking for get final product according to activity unit.Reagent should be preserved the way cryopreservation by biotechnological formulation.
Described human or animal's placenta amnion tissue is directly to obtain from human or animal body, animal again with Mammals for well.
The production process of placenta amnion cell extract of the present invention is preferably under the above-mentioned cold condition and carries out, and to reduce loss of activity, prevents to pollute.Cytoclasis should be complete, can adopt chemistry or mechanical means, technology such as ultrasonic wave, and it is broken fully that frozen-thaw process need proceed to cell repeatedly.How many protein contents can because of changing that salt solution adds, protein concn is preferably more than 1 mg/ml in the extracting solution, guarantee that other effective ingredient is able to abundant extraction, when being prepared into reagent, but reconcentration or be diluted to and require concentration, it is to contain the placenta amnion cell extract with 100 milliliters of nutrient solutions to represent with protein concn in application that activity defines, contain protein more than 1 milligram promptly the performance activity is arranged, be the effective active scope.Frozen is temperature requirement to freeze, and can preserve the activity of extract preferably, and long-pending amount back preparation reagent can be below-20 ℃, can prolonged preservation.Degerming method with filtration method for well.
Placenta amnion cell extract of the present invention is induced application in the differentiation at MSC, be with placenta amnion cell extract of the present invention, basic medium etc. and MSC co-cultivation, induce it to be divided into skin cells, its application can be that placenta amnion cell extract of the present invention is as the application of inductor in conjunction with prior art, also can be placenta amnion cell extract of the present invention and other reagent, become stem cell to induce the application of differentiation agents box as co-production such as basic medium, nutritive medium, the promoting growth of cell factor, antioxidant and microbiotic.The application of described test kit can be mainly by the application in the test kit of basic medium and ovum extract and the preparation of placenta amnion cell extract, or the application in the test kit of basic medium and placenta amnion cell extract preparation, described basic medium with the mixed culture medium that is selected from DMEM and F12 for well.
Using significant quantity is reactive conditions: represent with protein concn to contain the placenta amnion cell extract in the nutrient solution cumulative volume, be shown as promptly to show more than the protein 1% (mg/ml) activity is arranged, be the effective active scope, be effective active scope more at 1%-100%.In this field of activity, induce differentiation rate to reach more than 60%.
Through repetitious experiment, be example with people MSC respectively, amplification cultivation 15 days, go down to posterity four times, stem cell after the amplification is induced to differentiate into skin cells, cultivated again 15 days, go down to posterity four times, the differentiation rate of inducing that is induced to differentiate into skin cells reaches effect of the present invention, and repeatability and good stability.Inducing cell qualification result: have the skin cells form through microscopic examination, confirm to have the specific antigens sign of skin cells through immunocytochemistry.
Placenta amnion cell extract of the present invention is induced to differentiate in the application of skin cells at MSC, demonstrates the good differentiation rate of inducing, and more than 60%, suitable application induces differentiation rate to reach more than 95%.
Described contained composition of human or animal's placenta amnion cell extract and content thereof are unclear fully as yet at present, existing report confirms, contain the potential adjusting cell growth and the factor of breaking up in the placenta amnion cell cytosol, also contain rich in protein and correlation factor, for inducing differentiation, MSC provides ideal comprehensive nutrient environment jointly with other substratum and correlative factor, show as when keeping stem cell growth, induce it to skin cell differentiation.
Now the present invention is further elaborated in conjunction with the embodiments:
Embodiment 1, the preparation of placenta amnion cell extract
(1) get human or animal's placenta amnion tissue, clean with physiological saline, shred, adding equivalent physiological saline is standby;
(2) tissue homogenate or stamp mill be under 4 ℃ of conditions, ten thousand rev/mins of homogenate of 1-3 3-5 time, each 1 minute; Should notice that refiner is unsuitable overheated, otherwise portion of tissue liquid loses activity;
(3) homogenate is placed-20 ℃ of refrigerators frozen to freezing fully, general more than 24 hours, water proof thoroughly melts under 42 ℃ the condition not being higher than then, and as above the condition multigelation is 3-5 time, and smear microscopic examination routinely proves cell fragmentation fully;
(4) under 1-4 ℃ condition natural subsidence 2-10 hour, draw supernatant liquor, filter with 3-4 layer sterile gauze, place to go sediment and albumen floss, under 4 ℃ of conditions, centrifugal 30 minutes of 400-600g gets supernatant liquor;
(5) filters the supernatant liquor that obtains with filtering membrane under malleation or condition of negative pressure, molecular weight cut-off is greater than the macromolecular substance more than 200,000 dalton (Da), and the molecular weight that makes albumen in the filtered liquid and polypeptides matter is less than 200,000 dalton (Da)
(6) measuring protein concentration is 20 mg/ml, and application of active is measured and met the requirements, and the filtration method degerming is diluted to protein concentration with stroke-physiological saline solution and is 10 mg/ml, and is aseptic subpackaged, is aqua reagent.
Embodiment 2, and MSC is induced to differentiate into the application of skin cells
The application of test kit, ovum extract aqua contains the protein 10 mg/ml, and above-mentioned placenta amnion cell extract aqua contains the protein 10 mg/ml, prepares per 100 milliliters of test kits, each component independent packaging consumption:
(1) DMEM and F12 mixed culture medium are 80 milliliters
(2) ovum extract aqua is 10 milliliters
(3) placenta amnion cell extract aqua is 10 milliliters
The conventional promoting growth of cell factor, antioxidant and microbiotic etc. with significant quantity can be arranged in the test kit.
Method for inducing and cultivating:
1, mescenchymal stem cell separation, purifying: from tissues such as marrow, fat, separate obtaining mononuclearcell according to a conventional method, carry out earlier that the surperficial special sign antigen with mescenchymal stem cell carries out positive and negative immunoscreening after the cell adherent culture of former generation, obtain pure mescenchymal stem cell.If cell quantity is enough, also can directly carry out separation and purification with separating the mononuclearcell that obtains.According to experiment purpose and cell quantity, carry out cell amplification earlier and cultivate desired number or directly induce with this reagent.
2, mescenchymal stem cell adherent culture: is 1 * 10 with mescenchymal stem cell with the basic medium dilution
5Cell/ml is by 1 * 10
4Cell/cm
2Density is inoculated in the culturing bottle (ware), carries out vitro culture to cell by the mescenchymal stem cell amplification in vitro method and covers with a bottle wall, carries out inducing culture after the 2/3 above cytogamy.Mescenchymal stem cell criterion: form: fusiformis is the regular aligned growth of flamboyancy.Surface molecular sign: CD34
-, CD45
-, CD103
+, CD106
+, SH2
+, SH4
+
3, inducing culture: shift out the nutrient solution behind the adherent culture cell, wash 1-2 time with physiological saline, the cell of above-mentioned amount is induced, needing test kit nutrient solution cumulative volume be 10ML, and setting that two kinds of cell extract active protein content require respectively is 50%, then gets 9.0 milliliters of DMEM/F12 substratum, 0.5 milliliter of ovum extract, 0.5 milliliter of placenta amnion cell extract contains two kinds of each 5mg of cell extract protein in this 10ML nutrient solution, active protein concentration is respectively 50%.Other reagent such as the promoting growth of cell factor, antioxidant and microbiotic that can add significant quantity as required place 37 ℃, 5%CO2,95% humidity condition to cultivate down in the mixture of test kit nutrient solution and cell, continuously the observation of cell upgrowth situation.
4, inducing cell is identified:
(1) the distinctive Keratin sulfate of secretion skin cells, inducing cell form and skin cells are close, and relevant immunological testing meets the skin cells feature;
(2) inductivity: more than 90%;
(3) transplantation effect: be transplanted to human or animal's skin injury position, can repair skin injury with to retain cell well compatible.
Through a plurality of embodiment, contain the different protein concentrations of placenta amnion cell extract in 100 milliliters of nutrient solutions respectively and make inducing culture as 5%, 10%, 20%, 35%, 50%, 75%, 100% and 150%, all obtain satisfied effect, demonstrate good repeatability and stable.Replace the ovum extract with short skin cells somatomedin and nutritive medium etc., also obtain satisfied test effect.
Embodiment 3
Application in conjunction with prior art can be that existing external mescenchymal stem cell is induced to differentiate in the method for skin cells, and placenta amnion cell extract of the present invention is as the application of inducing reagent.
Technical scheme of the present invention is not restricted to the described embodiments.
Claims (10)
1. placenta amnion cell extract comprises that mainly following method produces:
(1) get human or animal's placenta amnion tissue, physiological saline is cleaned, and shreds standby;
(2) cell homogenates;
(3) the capable cytoclasis of homogenate is broken fully to cell;
(4) supernatant liquor is got in precipitation or filtration;
(5) separate the removal molecular weight greater than the above material of 200,000 dalton (Da);
(6) determination of protein concentration, determination of activity, degerming, frozen or the preparation reagent aseptic subpackaged, this is a placenta amnion cell extract of the present invention.
2. placenta amnion cell extract according to claim 1 is characterized in that, comprises that mainly following method produces:
(1) get human or animal's placenta amnion tissue, physiological saline is cleaned, and shreds standby;
(2) below 4 ℃, homogenate 3-5 time;
(3) method of cell disruption is freeze thawing, places refrigerator frozen to freezing fully homogenate, thoroughly melts not being higher than under 42 ℃ of conditions then, and as above the condition multigelation is 3-5 time, smear microscopic examination routinely, and cell is fragmentation fully;
(4) under 1-4 ℃ of condition, remove sediment and albumen floss, draw supernatant liquor;
(5) supernatant liquor of Huo Deing separate to be removed molecular weight greater than the above macromolecular substance of 200,000 dalton (Da), and the molecular weight that makes albumen in the filtered liquid of acquisition and polypeptides matter is less than 200,000 dalton (Da);
(6) the filtered liquid determining the protein quantity of Huo Deing, protein concn be more than 1 milligram/every milliliter of filtrate, determination of activity, and degerming, frozen or preparation reagent is aseptic subpackaged.
3. placenta amnion cell extract according to claim 1 and 2 is characterized in that, adds 1-4 times of physiological saline in the placenta amnion tissue of step (1) and dilutes homogenate again.
4. placenta amnion cell extract according to claim 1 and 2 is characterized in that, the separation method of step (5) is to filter under malleation or condition of negative pressure with filtering membrane.
5. placenta amnion cell extract according to claim 3 is characterized in that, the separation method of step (5) is to filter under malleation or condition of negative pressure with filtering membrane.
6. according to claim 1,2 or 5 described placenta amnion cell extracts, it is characterized in that described animal is a Mammals.
7. the placenta amnion cell extract of claim 1 is induced application in the differentiation at mescenchymal stem cell, is the application that external evoked mescenchymal stem cell is divided into skin cells.
8. application according to claim 7 is characterized in that, described application is the application in the preparation test kit.
9. application according to claim 8 is characterized in that, the application of described test kit is the application of the test kit of the basic medium mixed culture medium that is selected from DMEM and F12.
10. according to Claim 8 or 9 described application, it is characterized in that, the application of described test kit mainly is the application in the test kit of basic medium and ovum extract and the preparation of placenta amnion cell extract, or mainly is the application in the test kit of basic medium and the preparation of placenta amnion cell extract.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1948466B (en) * | 2006-11-10 | 2010-05-12 | 中国人民解放军军事医学科学院野战输血研究所 | Reagent box used for preparing bone marrow interstitial stem cell |
| CN101589137B (en) * | 2005-03-31 | 2013-06-05 | 斯丹姆涅恩有限公司 | Amnion-derived cell compositions, methods of making and uses thereof |
| CN103191155A (en) * | 2012-01-06 | 2013-07-10 | 上海交通大学医学院 | Application of mesenchymal stem cells in preparation of multiple sclerosis treatment medicines, and extraction method of mesenchymal stem cells |
| US8591883B2 (en) | 2005-12-29 | 2013-11-26 | Anthrogenesis Corporation | Placental stem cell populations |
| US8969315B2 (en) | 2010-12-31 | 2015-03-03 | Anthrogenesis Corporation | Enhancement of placental stem cell potency using modulatory RNA molecules |
| US9040035B2 (en) | 2011-06-01 | 2015-05-26 | Anthrogenesis Corporation | Treatment of pain using placental stem cells |
| CN108472241A (en) * | 2016-12-01 | 2018-08-31 | 荷鲁斯股份有限公司 | The manufacturing method of oil-soluble intacellin |
| US10104880B2 (en) | 2008-08-20 | 2018-10-23 | Celularity, Inc. | Cell composition and methods of making the same |
| CN108753876A (en) * | 2018-05-25 | 2018-11-06 | 华子昂 | Utilize the method for sheep placenta extracts enhancing stem cell surface receptor CXCR4 expression |
| CN109280641A (en) * | 2018-08-31 | 2019-01-29 | 华子昂 | Enhance the method that Derived from Mesenchymal Stem Cells is nerve cell |
| CN110373382A (en) * | 2019-06-12 | 2019-10-25 | 秦海燕 | A kind of extracting method of ips cell extract and reversed using ips cell extract aging mescenchymal stem cell method |
| CN111467292A (en) * | 2020-04-03 | 2020-07-31 | 焕生汇生物基因技术(北京)有限公司 | Preparation method of human placenta extract supernatant |
| CN111996163A (en) * | 2020-09-04 | 2020-11-27 | 安徽惠恩生物科技股份有限公司 | Skin anti-aging method based on mesenchymal stem cell disrupted cytoplasm |
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| CN101589137B (en) * | 2005-03-31 | 2013-06-05 | 斯丹姆涅恩有限公司 | Amnion-derived cell compositions, methods of making and uses thereof |
| US9078898B2 (en) | 2005-12-29 | 2015-07-14 | Anthrogenesis Corporation | Placental stem cell populations |
| US10383897B2 (en) | 2005-12-29 | 2019-08-20 | Celularity, Inc. | Placental stem cell populations |
| US8591883B2 (en) | 2005-12-29 | 2013-11-26 | Anthrogenesis Corporation | Placental stem cell populations |
| US8691217B2 (en) | 2005-12-29 | 2014-04-08 | Anthrogenesis Corporation | Placental stem cell populations |
| CN1948466B (en) * | 2006-11-10 | 2010-05-12 | 中国人民解放军军事医学科学院野战输血研究所 | Reagent box used for preparing bone marrow interstitial stem cell |
| US10104880B2 (en) | 2008-08-20 | 2018-10-23 | Celularity, Inc. | Cell composition and methods of making the same |
| US8969315B2 (en) | 2010-12-31 | 2015-03-03 | Anthrogenesis Corporation | Enhancement of placental stem cell potency using modulatory RNA molecules |
| US9040035B2 (en) | 2011-06-01 | 2015-05-26 | Anthrogenesis Corporation | Treatment of pain using placental stem cells |
| US11090339B2 (en) | 2011-06-01 | 2021-08-17 | Celularity Inc. | Treatment of pain using placental stem cells |
| CN103191155B (en) * | 2012-01-06 | 2015-04-01 | 上海交通大学医学院 | Application of mesenchymal stem cells in preparation of multiple sclerosis treatment medicines, and extraction method of mesenchymal stem cells |
| CN103191155A (en) * | 2012-01-06 | 2013-07-10 | 上海交通大学医学院 | Application of mesenchymal stem cells in preparation of multiple sclerosis treatment medicines, and extraction method of mesenchymal stem cells |
| CN108472241A (en) * | 2016-12-01 | 2018-08-31 | 荷鲁斯股份有限公司 | The manufacturing method of oil-soluble intacellin |
| CN108472241B (en) * | 2016-12-01 | 2021-02-09 | 荷鲁斯股份有限公司 | Method for preparing oil soluble placenta extract |
| CN108753876A (en) * | 2018-05-25 | 2018-11-06 | 华子昂 | Utilize the method for sheep placenta extracts enhancing stem cell surface receptor CXCR4 expression |
| CN108753876B (en) * | 2018-05-25 | 2022-06-14 | 华子昂 | Method for enhancing expression of stem cell surface receptor CXCR4 by using sheep placenta extract |
| CN109280641A (en) * | 2018-08-31 | 2019-01-29 | 华子昂 | Enhance the method that Derived from Mesenchymal Stem Cells is nerve cell |
| CN110373382A (en) * | 2019-06-12 | 2019-10-25 | 秦海燕 | A kind of extracting method of ips cell extract and reversed using ips cell extract aging mescenchymal stem cell method |
| CN111467292A (en) * | 2020-04-03 | 2020-07-31 | 焕生汇生物基因技术(北京)有限公司 | Preparation method of human placenta extract supernatant |
| CN111996163A (en) * | 2020-09-04 | 2020-11-27 | 安徽惠恩生物科技股份有限公司 | Skin anti-aging method based on mesenchymal stem cell disrupted cytoplasm |
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