Name: Forfatter Spotlight: Fremme hepatocyttrensing fra humant induserte pluripotente stamceller for regenerativ medisin
Uploaded: 2023-12-01T12:50:00
Description: Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

Dette arbeidet ble støttet av departementet for utdanning, kultur, sport, vitenskap og teknologi tilskuddsnummer [23390072].

Denne studien brukte kommersielt oppnådde humane iPS-celler (253G1-stamme) (se materialfortegnelse).
1. Vedlikehold av humane iPS-celler
<ol><li>Oppretthold humane iPS-celler under matefrie forhold i AK02-medium på kulturskåler belagt med 0,25 μg/cm2 iMatrix-511 (se materialtabell).</li>
</ol>2. Leverdifferensiering av humane iPS-celler i 2D-kulturer
MERK: Se <...

Rensing av humane iPSC-avledede hepatocytter ved mitokondriell-ALCAM-sortering

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På grunn av deres funksjoner i næringsmetabolisme og avgiftning, har hepatocytter et relativt stort antall mitokondrier sammenlignet med andre celletyper15. ALCAM er medlem av immunglobulin-superfamilien og spiller en rolle i celleadhesjon og migrasjon. Det kommer til uttrykk i ulike celletyper, inkludert lever-, epitel-, lymfocytiske, myeloid-, fibroblast- og nevronale celler16. Ved å bruke en kombinasjon av den mitokondriebaserte metoden og ALCAM-antistoffet, ble human...

Embryonale og induserte pluripotente stamceller (henholdsvis ES og iPS) anses som lovende cellekilder for regenerative terapier. Effektiviteten av å differensiere disse cellene til spesifikke målcelletyper kan imidlertid variere, selv når du bruker samme cellelinje, protokoll og eksperimentator 1,2,3,4. Denne variasjonen kan tilskrives den høye følsomheten til humane ES/iPS-celler for miljøet. Derfor er det for tiden vanskelig å konsekvent oppnå rene målceller. For å oppnå svært sikker regenerativ medisin er det avgjørende å eliminere proliferative celler og udifferensierte stamceller i terapeutiske celler, og avansert renseteknologi for målceller er avgjørende 5,6,7.
En cellesorterer er en enhet som øyeblikkelig analyserer individuelle celler og sorterer levende celler av interesse basert på de fluorescerende signalstyrkene, og tilbyr en lovende løsning. Dette kan oppnås gjennom antistofffarging av celletypespesifikke overflatemarkører eller ved å bruke celletypespesifikke reportergenuttrykk. Ved å bruke denne teknikken er det flere rapporter om metoder for å rense pluripotente stamcelleavledede kardiomyocytter 8,9,10 og hepatocytter11,12. Hattori et al. utviklet en innovativ mitokondriell rensemetode ved hjelp av en cellesorterer13. Ved å dra nytte av det faktum at kardiomyocytter har høye energibehov gjennom mitokondriell aktivitet, kan farging av cellene med det levende mitokondrie-indikative fargestoffet TMRM (tetrametylrhodaminmetylester) brukes til å merke og sterkt rense kardiomyocytter med FACS fra humane ES-celleavledede embryoidlegemer som inneholder forskjellige celletyper. Fraværet av tumorigenisitet ble bekreftet av teratomdannelsesanalyser med de rensede kardiomyocyttene. Videre oppdaget Yamashita et al. uventet en metode for å rense hepatocytter fra humane ES-celleavledede embryoidlegemer ved å isolere fraksjoner med høy mitokondriell aktivitet og ALCAM-positivt uttrykk14. Begrunnelsen for denne metoden er at hepatocytter også har et relativt høyt antall mitokondrier på grunn av deres høye forbruk av ATP for næringsmetabolisme og avgiftning15, og hepatocytter uttrykker ALCAM, et medlem av immunglobulin-superfamilien, som spiller en rolle i celleadhesjon og migrasjon16.
Tidligere høyrensende metoder for pluripotente stamcelleavledede hepatocytter krevde genetiske modifikasjoner, og ikke-genetiske rensemetoder hadde lav effektivitet17. Den mitokondrielle ikke-genetiske metoden har fortjeneste for å oppnå høy renhet. Når det er behov for leverstamceller, kan CD133- og CD13- eller Dlk1-baserte metoder18,19 velges. Selv om nøyaktigheten til genomredigeringsteknologi har avansert, kan den potensielle risikoen for uforutsette genomiske endringer (f.eks. karsinogenese) ikke elimineres helt. Metoder basert på mitokondriell aktivitet, uten å involvere genmodifisering, kan være fri for slike risikoer.
Som et resultat av undersøkelse av ulike mitokondrielle indikatorer i neonatale rottekardiomyocytter, ble TMRM observert å forsvinne helt innen 24 timer, mens andre fargestoffer forble i minst 5 dager13. Videre er det viktig å merke seg at TMRM og JC-1 ikke påvirket cellelevedyktighet når de ble vurdert med 3-(4,5-dimetyl-tiazol-2-yl)-2,5-difenyltetrazoliumbromid (MTT)-analysen, mens andre fargestoffer viste varierende effekter på cellelevedyktighet13. TMRM demonstrerer høyere sikkerhet.
Til dags dato har flere todimensjonale (2D) differensieringsmetoder blitt utviklet som mer effektive tilnærminger for å indusere differensiering sammenlignet med tredimensjonal (3D) embryoid kroppsdannelse. Dette er fordi trinnvise differensieringsinduserende forbindelser eller cytokiner kan administreres jevnt til celler på et 2D-plan, i stedet for i et 3D-rom. I denne studien ble den tidligere rapporterte metode20 modifisert for å indusere differensiering til humane iPS-celleavledede hepatocytter. Her er detaljene om prosedyrene for oppdatert 2D-differensiering og rensing av hepatocytter avledet fra humane iPS-celler beskrevet.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>253G1 human iPS cell line</td>
			<td>RIKEN BioResource Research Center</td>
			<td>HPS0002</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>163-20145</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A Solution, Human, Recombinant</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>18585</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nuclei Antibody, clone 235-1</td>
			<td>Chemicon</td>
			<td>MAB1281</td>
			<td>Antibody against human nuclear antigen</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>MedChemExpress</td>
			<td>HY-10182&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciclosporin A</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>035-18961</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>034-22363</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Gel-like basement membrane matrix</td>
		</tr>
		<tr>
			<td>CultureSure Y-27632</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>036-24023</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>047-18863</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>047-29353</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10036</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51820-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Dipeptide L-Alanyl-L-Glutamine</td>
		</tr>
		<tr>
			<td>Human/Mouse/Rat/Canine ALCAM/CD166 Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1172</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone 21-hemisuccinate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2270</td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 silk</td>
			<td>Nippi</td>
			<td>892021</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoBlock</td>
			<td>KAC</td>
			<td>CTKN001</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>ITS-G Supplement(&#215;100)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>090-06741</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>128-06075</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hexanoic-Try-Ile-(6)-amino Hexanoic amide (Dihexa)</td>
			<td>Toronto Research Chemicals</td>
			<td>H293745</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-Human Albumin</td>
			<td>Dako</td>
			<td>A0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene Sorbitan Monolaurate (Tween 20)</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>28353-85</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>189-02025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-Ascorbate</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>03422-32</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFit AK02N</td>
			<td>REPROCELL</td>
			<td>RCAK02N</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS (10x)</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>12748-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, methyl ester (TMRM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>T668</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>28229-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>20577-34</td>
			<td></td>
		</tr>
		<tr>
			<td>TRYPSIN 250</td>
			<td>Difco</td>
			<td>215240</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose phosphate broth solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8159</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of live mitochondria staining in cell-sorting to purify hepatocytes derived from human induced pluripotent stem cells

Humane embryonale stamceller (ES) og induserte pluripotente stamceller (iPS) har potensielle anvendelser i cellebasert regenerativ medisin for behandling av alvorlig syke organer på grunn av deres ubegrensede spredning og pluripotente egenskaper. Det er imidlertid utfordrende å differensiere humane ES/iPS-celler til 100 % rene målcelletyper på grunn av deres høye følsomhet for miljøet. Tumorgenese etter transplantasjon er forårsaket av forurensede, prolifererende og udifferensierte celler, noe som gjør høyrensingsteknologi avgjørende for sikker realisering av regenerativ medisin. For å redusere risikoen for tumorgenese er det utviklet en høyrensingsteknologi for humane iPS-celleavledede hepatocytter. Metoden bruker FACS (fluorescensaktivert cellesortering) ved bruk av en kombinasjon av høyt mitokondrieinnhold og celleoverflatemarkøren ALCAM (aktivert leukocyttcelleadhesjonsmolekyl) uten genetisk modifikasjon. 97 % ± 0,38 % (n = 5) av de rensede hepatocyttene ved bruk av denne metoden viste albuminproteinekspresjon. Denne artikkelen tar sikte på å gi detaljerte prosedyrer for denne metoden, slik den brukes på den nyeste todimensjonale differensieringsmetoden for humane iPS-celler til hepatocytter.

Embryonic and induced pluripotent stem cells (ES and iPS, respectively) are considered promising cell sources for regenerative therapies. However, the efficiency of differentiating these cells into specific target cell types can vary, even when using the same cell line, protocol, and experimenter1,2,3,4. This variability may be attributed to the high sensitivity of human ES/iPS cells to their environment. Therefore, it is currently difficult to consistently obtain pure target cells. To achieve highly safe regenerative medicine, it is crucial to eliminate proliferative cells and undifferentiated stem cells in therapeutic cells, and advanced purification technology for target cells is essential5,6,7.
A cell sorter is a device that instantaneously analyzes individual cells and sorts live cells of interest based on the fluorescent signal strengths, offering a promising solution. This can be accomplished through antibody staining of cell type-specific surface markers or by utilizing cell type-specific reporter gene expressions. Using this technique, there are several reports on methods for purifying pluripotent stem cell-derived cardiomyocytes8,9,10 and hepatocytes11,12. Hattori et al. developed an innovative mitochondrial purification method using a cell sorter13. Taking advantage of the fact that cardiomyocytes have high energy demands through mitochondrial activity, staining the cells with the live mitochondria-indicative dye TMRM (tetramethylrhodamine methyl ester) can be used to label and highly purify cardiomyocytes by FACS from human ES cell-derived embryoid bodies containing various cell types. The absence of tumorigenicity was confirmed by teratoma formation assays with the purified cardiomyocytes. Furthermore, Yamashita et al. unexpectedly discovered a method to purify hepatocytes from human ES cell-derived embryoid bodies by isolating fractions with high mitochondrial activity and ALCAM-positive expression14. The rationale for this method is that hepatocytes also have a relatively high number of mitochondria due to their high consumption of ATP for nutrient metabolism and detoxification15, and hepatocytes express ALCAM, a member of the immunoglobulin superfamily, which plays a role in cell adhesion and migration16.
Previous highly purifying methods for pluripotent stem cell-derived hepatocytes required genetic modifications, and non-genetic purification methods had low efficiency17. The mitochondrial non-genetic method holds merit for achieving high purity. When hepatic progenitor cells are needed, CD133 and CD13- or Dlk1-based methods18,19 can be chosen. Although the accuracy of genome editing technology has advanced, the potential risk of unforeseen genomic changes (e.g., carcinogenesis) cannot be entirely eliminated. Methods based on mitochondrial activity, without involving genetic modification, can be free from such risks.
As a result of examining various mitochondrial indicators in neonatal rat cardiomyocytes, TMRM was observed to disappear completely within 24 h, whereas other dyes remained for at least 5 days13. Moreover, it is important to note that TMRM and JC-1 did not impact cell viability when assessed with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while other dyes demonstrated varying effects on cell viability13. TMRM demonstrates higher safety.
To date, several two-dimensional (2D) differentiation methods have been developed as more efficient approaches for inducing differentiation compared to three-dimensional (3D) embryoid body formation. This is because step-wise differentiation-inducing compounds or cytokines can be uniformly administered to cells on a 2D plane, rather than in a 3D space. In this study, the previously reported method20 was modified to induce differentiation into human iPS cell-derived hepatocytes. Here, the details of the procedures for the up-to-date 2D differentiation and purification of hepatocytes derived from human iPS cells are described.

Forfatter Spotlight: Fremme hepatocyttrensing fra humant induserte pluripotente stamceller for regenerativ medisin

Påføring av levende mitokondrierfarging i cellesortering for å rense hepatocytter avledet fra humant induserte pluripotente stamceller

The scope of our research is to purify human induced pluripotent stem cell-derived hepatocytes, using without genetic modification. We aim to address the issue of the tumorigenic risk associated with the undifferentiated cells, among those differentiated from human iPS cells. One of the current experimental challenges is a low cell purity, due to the mechanical storage of sorting.We are developing a new purification method, that utilizes the unique metabolism features of hepatocytes, for large-scale purification. One non-cell sorting method for hepatic progenitor cells cannot obtain pure hepatocytes. Another one for mature hepatocytes can only treat a small fraction of hepatocytes, because of the limited expression of the marker protein.In contrast, our approach can obtain hepatocytes at different maturation levels with a higher eve. Human iPS cell-derived hepatocytes are extremely immature, and show more reduced hepatocyte macro expression after purification. Our future goal is to enhance hepatocyte modulation using organoidal technology and day-to-day advancements to regenerative medicine and drug discovery.

Tidslinjen for prosessene som induserer humane iPS-celler til å differensiere til hepatocytter gjennom 2D-kultur (figur 1A) og representative celletrekk (figur 1B) er vist. Omtrent på differensieringsdag 12 begynte cellene å vise polygonale celleformer og runde kjerner, karakteristisk for hepatocytter. Noen hepatocytter viste også multinukleasjon.
FACS-analyse ble utført ved bruk av cellene på differensieringsdag 27. For å iden...

Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

Hepatocytter avledet fra pluripotente stamceller kan renses gjennom cellesortering, ved bruk av en kombinasjon av mitokondrielt og aktivert leukocyttcelleadhesjonsmolekyl (ALCAM, også kjent som CD166) farging.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

Omfanget av vår forskning er å rense humant induserte pluripotente stamcelle-avledede hepatocytter, ved bruk uten genetisk modifikasjon. Vi tar sikte på å ta opp spørsmålet om den tumorogene risikoen forbundet med de udifferensierte cellene, blant de som er differensiert fra humane iPS-celler. En av de nåværende eksperimentelle utfordringene er en lav cellerenhet, på grunn av den mekaniske lagringen av sortering.Vi utvikler en ny rensemetode, som utnytter de unike metabolismeegenskapene til hepatocytter, for storskala rensing. En ikke-cellesorteringsmetode for leverstamceller kan ikke oppnå rene hepatocytter. En annen for modne hepatocytter kan bare behandle en liten brøkdel av hepatocytter, på grunn av det begrensede uttrykket av markørproteinet.Derimot kan vår tilnærming oppnå hepatocytter på forskjellige modningsnivåer med en høyere eve. Humane iPS-celleavledede hepatocytter er ekstremt umodne, og viser mer redusert hepatocyttmakrouttrykk etter rensing. Vårt fremtidige mål er å forbedre hepatocyttmodulering ved hjelp av organoidal teknologi og daglige fremskritt innen regenerativ medisin og legemiddeloppdagelse.

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Research

JoVE Journal

Biology

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

Hepatic Differentiation of Human-Induced Pluripotent Stem Cells in 2D Cultures

To begin, coat each well of a six-well plate with one milliliter of basement membrane matrix and incubate at 37 degrees Celsius for one hour. Remove the coated matrix from the plate and add human-induced pluripotent stem cells prepared in AK02N medium supplemented with 10 micromolar ROCK inhibitor. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for three days.After three days, on day zero of the initiating hepatic differentiation, wash the cells once with RPMI 1640 medium. Next, to initiate endoderm differentiation, add RPMI B27 GlutaMAX supplemented with three to six micromolar CHIR 99021 and 100 nanograms per milliliter Activin A into the plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.On day one, replace the existing medium with RPMI B27 GlutaMAX supplemented with 50 nanograms per milliliter of Activin A.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. On day two, to induce hepatic progenitor differentiation, replace the existing medium with RPMI B27 GlutaMAX medium supplemented with 1%dimethyl sulfoxide, and continue incubation for up to five days. On day seven, to initiate hepatocyte maturation, switch to hepatocyte maturation medium and incubate for 20 days at 37 degrees Celsius and 5%carbon dioxide.On day 27, human-induced pluripotent stem cells displayed polygonal cell shapes and rounded nuclei, characteristics of hepatocytes.

Leverdifferensiering av menneskeinduserte pluripotente stamceller i 2D-kulturer

Cell Preparation for Purification of Human-Induced Pluripotent Stem Cells-Derived Hepatocytes

After 27 days of initiating hepatic differentiation of human induced pluripotent stem cells, wash the cells with two milliliters of PBS per well. Prepare the enzyme solution in ADS buffer using the given components. Add one milliliter of solution per well of a six well plate.Place the plate at 37 degrees Celsius on a rotating shaker for about two hours to detach the cells from the plate. Confirm the cell detachment under a microscope, then pipette the cell suspension to disperse into single cells, and collect them in a 50 milliliter tube containing the hepatocyte maturation medium. Centrifuge the cell suspension at 200G for five minutes at 18 degrees Celsius.And using an aspirator, remove the supernatant. To stain the mitochondria of cells, resuspend the pellet in five milliliters of hepatocyte maturation medium containing 100 nanomolar TMRM. Incubate the cells for 30 minutes at 37 degrees Celsius while protecting them from light.Again, centrifuge the cell suspension and resuspend the pellet in one to five milliliters of cold ADS buffer containing 2%FBS. After cell counting, aliquot 500, 000 cells to a 1.5 milliliter tube and label it as the no primary antibody control. Centrifuge the remaining cell suspension at 200G for five minutes at four degrees Celsius, and remove the supernatant.Add 100 microliters of diluted primary antibody per one million cells. Transfer the cell suspension to a 1.5 milliliter tube and incubate on ice for 50 minutes. After incubation, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS.Now add 100 microliters of diluted secondary antibody per one million cells to the primary antibody, stained or unstained cells, and incubate for 30 minutes on ice. Again, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS. After resuspending the cells in ADS buffer, filter them through a snap cap cell strainer and place the tube on ice.

Celleforberedelse for rensing av menneskeinduserte pluripotente stamceller-avledede hepatocytter

Fluorescence-Activated Cell Sorting of Hepatocytes Derived from Human Induced Pluripotent Stem Cells

To begin, set up the fluorescence-activated cell sorting, or FACS machine, for cell sorting following the manufacturer's instructions. Place the sample tube on the FACS machine and load it. Gate TMRM-high and ALCAM negative population in the P1 region to sort non-hepatocytes.Then gate the TMRM high and ALCAM positive population in the P2 region to sort hepatocytes. Collect sorted cells in 15-milliliter collection tubes containing hepatocyte maturation medium. After sorting, remove the collection tube from the FACS machine.Centrifuge the sorted cell suspension at 200 G for five minutes at 18 degrees Celsius. Remove the supernatant, and re-suspend the pellet in two milliliters of hepatocyte maturation medium with 10 micromolar rock inhibitor and 20 nanomolar cyclosporine A.Seed the cells on mitomycin C-treated mouse embryonic fibroblasts with hepatocyte maturation medium in a six-well plate. Culture the cells in a 37-degree Celsius carbon dioxide incubator for five days before immuno staining for hepatocyte purity test.FACS analysis conducted on day 27 of hepatic differentiation revealed TMRM high and ALCAM positive population. The immunohistochemical staining of P1 and P2 cells cultured on mouse embryonic fibroblasts after sorting is shown. A purity test by counting albumin-positive cells showed 0%of P1, and approximately 97%of P2 cells were hepatocyte-like cells.