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Application‌‌Protocol‌ ‌‌Maintenance‌‌of‌‌hPSC‌‌culture‌‌suitable‌‌for‌‌Quick-Tissue™‌‌Differentiation‌‌Kits‌ ‌Introduction‌ ‌‌The‌ ‌Quick-Tissue™‌ ‌Differentiation‌ ‌kits‌ ‌give‌ ‌the‌ ‌best‌ ‌differentiation‌ ‌results‌ ‌when‌ ‌source‌ ‌human‌ ‌pluripotent‌ ‌stem‌ ‌cells‌‌(hPSCs)‌‌have‌‌been‌‌maintained‌‌in‌‌StemFit®‌‌Basic04,‌‌StemFlex™‌‌Medium,‌‌or‌‌other‌‌similar‌‌culture‌‌media‌‌which‌‌enable‌‌the‌‌maintenance‌ ‌of‌ ‌cultures‌ ‌by‌ ‌single-cell‌ ‌passaging.‌ ‌This‌‌Application‌‌Protocol‌‌describes‌‌the‌‌method‌‌of‌‌maintaining‌‌hPSCs‌‌
under‌‌StemFit®‌‌Basic04‌‌conditions‌‌and‌‌addresses‌‌common‌‌pitfalls‌‌to‌‌be‌‌avoided.‌ ‌‌Required‌‌Consumables‌ ‌‌Item‌ ‌Vendor‌ ‌Catalog‌‌Number‌ ‌StemFit®‌‌Basic04‌‌(Complete‌‌type)‌ ‌Elixirgen‌‌Scientific‌ ‌ASB04-C‌ ‌iMatrix-511‌‌silk‌ ‌Elixirgen‌‌Scientific‌ ‌NI511S‌ ‌ROCK‌‌inhibitor‌‌Y27632‌ ‌Selleckchem‌ ‌S1049‌ ‌TrypLE‌‌Select‌‌Enzyme‌‌(1X)*‌ ‌ThermoFisher‌

12563011‌ ‌0.02%‌‌EDTA‌‌in‌‌DPBS*‌ ‌Sigma-Aldrich‌ ‌E8008-100ML‌ ‌Penicillin-Streptomycin‌ ‌ThermoFisher‌

15140122‌ ‌Phosphate-buffered‌‌saline‌‌(without‌‌Ca‌++‌‌ ‌Mg‌++‌)‌ ‌ThermoFisher‌

20012050‌ ‌Dimethyl‌‌sulfoxide‌‌(DMSO)‌ ‌Sigma-Aldrich‌ ‌D8418‌ ‌AMSBIO‌ ‌11890‌ ‌(Optional)‌‌STEM-CELLBANKER**‌ ‌‌*‌Can‌‌be‌‌substituted‌‌with‌‌our‌‌Cell‌‌Dissociation‌‌Reagent‌‌(Solution‌‌D1),‌‌Catalog‌‌Number:‌‌CDR.‌ ‌‌**‌This‌‌is‌‌only‌‌required‌‌if‌‌you‌‌intend‌‌to‌‌cryopreserve‌‌the‌‌cells.‌ ‌‌Condition‌‌of‌‌Use‌ ‌‌This‌‌product‌‌is‌‌for‌‌research‌‌use‌‌only.‌‌It‌‌is‌‌not‌‌approved‌‌for‌‌use‌‌in‌‌humans‌‌or‌‌for‌‌therapeutic‌‌or‌‌diagnostic‌‌use.‌ ‌‌‌Technical‌‌Support‌ ‌‌For‌‌technical‌‌support,‌‌please‌‌contact‌‌us‌‌at‌c‌ s@elixirgensci.com‌‌‌or‌‌call‌‌+1‌‌(443)‌‌869-5420‌‌(M-F‌‌9am-5pm‌‌EST).‌ ‌‌Last‌‌revised:‌‌October‌‌26,‌‌2021‌ ‌elixirgensci.com‌ ‌‌

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Workflow‌ ‌‌‌‌Media‌‌Preparation‌ ‌StemFit‌ ‌1.

Thaw‌‌StemFit®‌‌Basic04‌‌(Complete‌‌type)‌‌medium‌‌bottle‌‌at‌‌4°C‌‌for‌‌approximately‌‌48‌‌hours‌‌until‌‌thawed.‌ ‌Note:‌ ‌DO‌ ‌NOT‌ ‌warm‌ ‌the‌ ‌medium‌ ‌at‌ ‌37°C‌ ‌as‌ ‌it‌ ‌will‌ ‌result‌ ‌in‌ ‌degradation‌ ‌of‌ ‌medium‌ ‌components.‌ ‌See‌ ‌the‌‌manufacturer’s‌‌instructions‌‌for‌‌more‌‌information.‌ ‌2. Make‌‌aliquots‌‌of‌‌a‌‌convenient‌‌volume‌‌(e.g.,‌‌40‌‌ml).‌ ‌3. This‌‌solution,‌‌hereafter‌‌referred‌‌to‌‌as‌‌StemFit,‌‌can‌‌be‌‌stored‌‌at‌‌- 20°C‌‌to‌‌-80°C.‌‌Once‌‌thawed,‌‌StemFit‌‌should‌‌be‌‌stored‌‌at‌‌4°C‌‌for‌‌up‌‌to‌‌2‌‌weeks.‌ ‌10‌‌mM‌‌ROCK‌‌inhibitor‌‌Y27632‌‌(iROCK)‌ ‌1. Dissolve‌‌10‌‌mg‌‌ROCK‌‌inhibitor‌‌Y27632‌‌in‌‌3.12‌‌ml‌‌DMSO.‌ ‌2. Make‌‌aliquots‌‌of‌‌a‌‌convenient‌‌volume‌‌(e.g.,‌‌100‌‌μl).‌ ‌3. This‌‌solution,‌‌hereafter‌‌referred‌‌to‌‌as‌‌iROCK,‌‌can‌‌be‌‌stored‌‌at‌‌- 20°C.‌ ‌0.5X‌‌TrypLE‌‌Select‌‌with‌‌EDTA‌‌(Solution‌‌D1)*‌‌ ‌1. Mix‌‌1‌‌ml‌‌TrypLE‌‌Select‌‌Enzyme‌‌(1X)‌‌with‌‌1‌‌ml‌‌0.02%‌‌EDTA‌‌in‌‌DPBS.‌ ‌2. This‌m‌ ixture,‌‌hereafter‌‌referred‌‌to‌‌as‌‌Solution‌‌D1,‌‌can‌‌be‌‌stored‌‌at‌‌4°C‌‌for‌‌2‌‌weeks.‌ ‌*‌Can‌‌be‌‌substituted‌‌with‌‌our‌‌Cell‌‌Dissociation‌‌Reagent‌‌(Solution‌‌D1),‌‌Catalog‌‌Number:‌‌CDR.‌ ‌Notes‌ ‌●●●●In‌‌this‌‌protocol,‌‌instructions‌‌are‌‌described‌‌for‌‌one‌‌well‌‌of‌‌a‌‌6-well‌‌plate.‌‌For‌‌other‌‌well‌‌sizes,‌‌multiply‌‌the‌‌protocol’s‌‌
consumables‌‌by‌‌the‌‌ratio‌‌between‌‌well‌‌surface‌‌area‌‌sizes.‌ ‌This‌‌protocol‌‌assumes‌‌starting‌‌on‌‌a‌‌Monday‌‌or‌‌Tuesday‌‌so‌‌as‌‌to‌‌allow‌‌weekend‌‌free‌‌culture.‌ ‌Do‌‌not‌‌allow‌‌the‌‌cultures‌‌to‌‌become‌‌fully‌‌confluent.‌ ‌Spontaneously‌ ‌differentiating‌ ‌colonies‌ ‌can‌ ‌be‌‌manually‌‌removed.‌‌If‌‌a‌‌culture‌‌has‌‌more‌‌than‌‌20%‌‌spontaneously‌‌
differentiating‌‌colonies‌‌we‌‌recommend‌‌discarding‌‌the‌‌culture‌‌and‌‌starting‌‌over.‌ ‌‌elixirgensci.com‌ ‌‌

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Day‌‌0‌ ‌-4‌‌hours‌ ‌Note:‌ ‌Instead‌ ‌of‌‌precoating‌‌plates,‌‌many‌‌hPSC‌‌lines‌‌can‌‌be‌‌plated‌‌with‌‌iMatrix-511‌‌silk‌‌mixed‌‌into‌‌the‌‌cell‌‌suspension,‌‌a‌‌method‌‌referred‌‌to‌‌as‌‌“direct‌‌plating”.‌‌However,‌‌not‌‌all‌‌hPSC‌‌lines‌‌can‌‌be‌‌plated‌‌in‌‌this‌‌manner.‌‌It‌‌is‌‌recommended‌‌to‌‌seed‌‌
hPSCs‌ ‌on‌‌a‌‌precoated‌‌plate‌‌first‌‌and‌‌then‌‌test‌‌the‌‌direct‌‌plating‌‌method‌‌once‌‌the‌‌culture‌‌is‌‌maintained‌‌successfully‌‌(see‌‌
Appendix‌‌B).‌ ‌‌‌Plate‌‌preparation‌ ‌‌
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Prepare‌‌diluted‌‌iMatrix-511‌‌silk‌‌by‌‌mixing‌‌together‌‌the‌‌following‌‌components‌‌in‌‌a‌‌15‌‌ml‌‌conical‌‌tube.‌ ‌○ Make‌‌sure‌‌chilled‌‌PBS‌‌is‌‌used‌‌for‌‌this‌‌mixture.‌ ‌○ The‌‌leftover‌‌iMatrix-511‌‌silk‌‌can‌‌be‌‌stored‌‌at‌‌4°C‌‌for‌‌later‌‌uses.‌ ‌Diluted‌‌iMatrix-511‌‌silk‌‌Reagents‌ ‌Volume‌ ‌iMatrix-511‌‌silk‌ ‌4.8‌‌µl‌ ‌Chilled‌‌PBS‌ ‌2‌‌ml‌ ‌Add‌‌2‌‌ml‌‌diluted‌‌iMatrix-511‌‌silk‌‌to‌‌1‌‌well‌‌of‌‌a‌ ‌6-well‌‌plate.‌ ‌Incubate‌‌the‌‌plate‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌‌ ‌for‌‌2‌‌hours‌‌(or‌‌4°C‌‌overnight‌‌one‌‌day‌‌before‌‌Day‌‌1).‌ ‌After‌‌incubation,‌‌aspirate‌‌the‌‌supernatant‌‌from‌‌the‌‌well‌‌and‌‌add‌‌2‌‌ml‌‌PBS.‌ ‌Incubate‌‌the‌‌plate‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌‌ ‌until‌‌hPSCs‌‌are‌‌ready‌‌for‌‌plating.‌ ‌‌Passaging‌ ‌‌
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9.

Thaw‌ ‌an‌ ‌aliquot‌ ‌of‌ ‌StemFit‌ ‌at‌ ‌4°C‌ ‌overnight‌ ‌or‌ ‌warm‌ ‌it‌ ‌at‌ ‌room‌ ‌temperature,‌ ‌protected‌ ‌from‌ ‌light,‌ ‌for‌ ‌20-30‌‌
minutes.‌ ‌Warm‌‌Solution‌‌D1‌‌at‌‌room‌‌temperature‌‌for‌‌20-30‌‌minutes.‌‌ ‌Transfer‌‌4‌‌ml‌‌StemFit‌‌into‌‌a‌‌tube‌‌and‌‌add‌‌4‌‌μl‌‌10‌‌mM‌‌iROCK.‌‌Mix‌‌well.‌‌The‌‌resulting‌‌medium‌‌is‌‌hereafter‌ ‌referred‌‌to‌‌as‌‌iStemFit.‌ ‌Aspirate‌‌old‌‌medium‌‌from‌‌the‌‌culture‌‌and‌‌add‌‌1.5‌‌ml‌‌iStemFit.‌ ‌Incubate‌‌the‌‌culture‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌‌ ‌for‌‌1‌‌hour.‌‌This‌‌treatment‌‌enhances‌‌the‌‌survival‌‌of‌‌hPSCs‌‌after‌‌passaging.‌ ‌Tap‌‌the‌‌Solution‌‌D1‌‌tube‌‌5‌‌times‌‌with‌‌a‌‌finger‌‌and‌‌centrifuge‌‌at‌‌maximum‌‌speed‌‌for‌‌1‌‌minute.‌ ‌Aspirate‌‌iStemFit‌‌from‌‌the‌‌hPSC‌‌culture‌‌and‌‌add‌‌2‌‌ml‌‌PBS.‌ ‌Rock‌‌the‌‌plate‌‌3‌‌times,‌‌aspirate‌‌PBS‌‌from‌‌the‌‌culture,‌‌and‌‌add‌‌300‌‌μl‌‌Solution‌‌D1.‌ ‌Incubate‌‌the‌‌culture‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌ ‌for‌‌5‌‌minutes.‌‌If‌‌all‌‌the‌‌cells‌‌are‌‌not‌‌rounded‌‌under‌‌a‌‌microscope,‌‌continue‌‌to‌‌incubate‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌‌ ‌in‌‌1 -2‌‌minute‌‌increments‌‌(see‌‌images‌‌below).‌ ‌‌‌10. Carefully‌‌pipet‌‌out‌‌Solution‌‌D1‌‌from‌‌the‌‌culture‌‌using‌‌a‌‌P1000‌‌pipettor‌‌and‌‌add‌‌1‌‌ml‌‌iStemFit.‌ ‌11. Disperse‌‌the‌‌medium‌‌over‌‌the‌‌bottom‌‌surface‌‌of‌‌the‌‌well‌‌by‌‌pipetting‌‌8-15‌‌times‌‌to‌‌detach‌‌cells.‌ ‌12. Using‌‌the‌‌same‌‌pipette‌‌tip,‌‌collect‌‌the‌‌cell‌‌suspension‌‌in‌‌a‌‌1.5‌‌ml‌‌tube.‌‌ ‌‌elixirgensci.com‌ ‌‌3‌‌

13. Count‌‌cells‌‌to‌‌determine‌‌the‌‌volume‌‌of‌‌cell‌‌suspension‌‌needed.‌ ‌Note:‌‌The‌‌optimal‌‌seeding‌‌density‌‌will‌‌depend‌‌on‌‌the‌‌cell‌‌line‌‌being‌‌used‌‌and‌‌the‌‌desired‌‌time‌‌until‌‌next‌‌passage.‌‌We‌‌typically‌‌plate‌‌0.5-1‌‌x‌‌10‌4‌‌ ‌cells‌‌per‌‌well‌‌for‌‌an‌‌8-day‌‌culture‌‌schedule.‌‌ ‌14. Take‌‌out‌‌the‌‌determined‌‌volume‌‌of‌‌the‌‌cell‌‌suspension‌‌from‌‌the‌‌previous‌‌step,‌‌place‌‌it‌‌in‌‌a‌‌new‌‌tube‌‌and‌‌bring‌‌the‌‌volume‌‌up‌‌to‌‌1.5‌‌ml‌‌with‌‌iStemFit.‌ ‌15. Aspirate‌‌PBS‌‌from‌‌a‌‌new‌‌iMatrix-511‌‌silk-coated‌‌well‌‌and‌‌add‌‌1.5‌‌ml‌‌cell‌‌suspension.‌ ‌Note:‌‌The‌‌rest‌‌of‌‌the‌‌cells‌‌can‌‌be‌‌used‌‌for‌‌assays‌‌or‌‌frozen‌‌down‌‌following‌‌the‌‌instructions‌‌in‌‌Appendix‌‌C.‌ ‌16. Incubate‌‌the‌‌culture‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌‌ ‌for‌‌2‌‌days.‌ ‌‌Day‌‌2-6‌ ‌<‌‌1‌‌hour‌ ‌Medium‌‌Change‌ ‌
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Warm‌‌StemFit‌‌for‌‌20-30‌‌minutes‌‌at‌‌room‌‌temperature.‌ ‌Aspirate‌‌the‌‌old‌‌medium‌‌from‌‌the‌‌well‌‌and‌‌add‌‌1.5‌‌-‌‌4.5‌‌ml‌‌StemFit‌‌(See‌‌Notes‌‌below).‌ ‌Incubate‌‌the‌‌culture‌‌at‌‌37°C,‌‌5%‌‌CO‌2‌.‌ ‌Repeat‌‌steps‌‌1 -3‌‌daily‌‌until‌‌Day‌‌7‌‌(See‌‌Notes‌‌below).‌ ‌Notes:‌ ‌● When‌‌at‌‌low‌‌confluence‌‌shortly‌‌after‌‌passage,‌‌wells‌‌can‌‌be‌‌fed‌‌with‌‌1.5‌‌ml‌‌of‌‌medium.‌‌The‌‌volume‌‌required‌‌
will‌‌increase‌‌as‌‌the‌‌cultures‌‌become‌‌more‌‌confluent.‌ ‌● For‌‌weekend-free‌‌culturing,‌‌feed‌‌the‌‌well‌‌with‌‌4.5‌‌ml‌‌of‌‌medium‌‌on‌‌Friday‌‌afternoon.‌‌Cultures‌‌can‌‌then‌‌be‌‌left‌‌until‌‌Monday‌‌morning.‌‌ ‌● If‌ ‌the‌ ‌color‌ ‌of‌‌the‌‌culture‌‌medium‌‌becomes‌‌orange‌‌or‌‌yellow‌‌the‌‌medium‌‌should‌‌be‌‌changed‌‌daily‌‌with‌‌a‌‌larger‌‌volume‌‌of‌‌2-3‌‌ml.‌‌ ‌● Do‌ ‌not‌ ‌allow‌ ‌the‌ ‌cultures‌ ‌to‌ ‌become‌ ‌fully‌ ‌confluent.‌ ‌Passage‌ ‌as‌ ‌needed‌ ‌when‌ ‌approaching‌ ‌80%‌‌
confluency.‌ ‌‌Day‌‌7‌ ‌<‌‌1‌‌hour‌ ‌CONTINUOUS‌‌CULTURE‌‌OR‌‌FREEZING‌‌ ‌On‌‌Day‌‌7‌‌cultures‌‌should‌‌be‌‌ready‌‌for‌‌passaging‌‌again‌‌or‌‌freezing.‌ ‌‌‌elixirgensci.com‌ ‌‌

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Appendix‌‌A‌ ‌‌‌Figure‌‌1.‌‌‌Representative‌‌4x‌‌(left)‌‌and‌‌10x‌‌(right)‌‌phase‌‌contrast‌‌images‌‌of‌‌human‌‌iPSC‌‌cultures‌‌(scale‌‌bars‌‌=‌‌100‌‌μm).‌‌(A)‌‌Images‌‌were‌‌taken‌‌on‌‌day‌‌1‌‌after‌‌plating‌‌and‌‌show‌‌that‌‌cultures‌‌contain‌‌single‌‌cells‌‌or‌‌very‌‌small‌‌clusters‌‌of‌‌less‌‌than‌‌10‌‌cells.‌‌(B)‌‌Images‌‌were‌‌taken‌‌on‌ ‌day‌ ‌7‌‌after‌‌plating‌‌and‌‌show‌‌that‌‌cultures‌‌contain‌‌large‌‌distinct‌‌colonies‌‌with‌‌smooth‌‌edges‌‌and‌‌dense‌‌centers.‌‌These‌‌cultures‌‌are‌‌ready‌‌for‌‌passaging‌‌or‌‌freezing.‌‌(C)‌‌Images‌‌of‌‌cultures‌‌that‌‌are‌‌over-confluent‌‌with‌‌many‌‌colonies‌‌touching‌‌and‌‌merging‌‌together.‌ ‌‌‌‌Appendix‌‌B‌ ‌Direct‌‌Coating‌‌Method‌ ‌To‌ ‌culture‌ ‌hPSC‌ ‌using‌ ‌direct‌ ‌coating,‌ ‌skip‌ ‌precoating‌ ‌the‌ ‌wells‌ ‌with‌ ‌diluted‌ ‌iMatrix-511‌ ‌silk‌ ‌as‌ ‌instructed‌ ‌in‌ ‌the‌ ‌“plate‌‌
preparation”‌ ‌step.‌ ‌Instead,‌ ‌add‌ ‌4‌ ‌μl‌ ‌per‌ ‌well‌ ‌iMatrix-511‌ ‌silk‌ ‌to‌‌the‌‌harvested‌‌cell‌‌suspension‌‌immediately‌‌before‌‌plating‌‌‌
elixirgensci.com‌ ‌‌5‌‌during‌‌step‌‌14‌‌in‌‌the‌‌“passaging”‌‌section.‌‌Mix‌‌by‌‌pipetting‌‌up‌‌and‌‌down‌‌and‌‌dispense‌‌cells‌‌with‌‌iMatrix-511‌‌silk‌‌into‌‌each‌‌well.‌‌ ‌‌Note:‌ ‌Once‌ ‌iMatrix-511‌ ‌silk‌ ‌is‌ ‌added‌ ‌to‌ ‌the‌ ‌master‌‌mix,‌‌work‌‌as‌‌quickly‌‌as‌‌possible‌‌otherwise‌‌the‌‌plating‌‌efficiency‌‌may‌‌decrease.‌‌Keep‌‌in‌‌mind‌‌that‌‌iMatrix-511‌‌silk‌‌becomes‌‌solidified‌‌when‌‌it‌‌is‌‌left‌‌at‌‌15°C‌‌or‌‌above‌‌for‌‌5 -10‌‌minutes.‌‌ ‌‌‌‌Appendix‌‌C‌ ‌Freezing‌‌cells‌‌down‌ ‌Note:‌‌After‌‌thawing‌‌frozen‌‌cells,‌‌over‌‌60%‌‌of‌‌the‌‌cells‌‌will‌‌be‌‌viable.‌ ‌‌1. Determine‌‌the‌‌volume‌‌of‌‌the‌‌cell‌‌suspension‌‌and‌‌number‌‌of‌‌1.5‌‌or‌‌2.0‌‌ml‌‌cryovials‌‌needed‌‌to‌‌freeze‌‌2.5‌‌x‌‌10‌5‌ ‌cells‌‌
per‌‌cryovial.‌ ‌2. Centrifuge‌‌the‌‌cell‌‌suspension‌‌at‌‌200‌‌x‌‌g‌‌for‌‌4‌‌minutes.‌‌ ‌3. While‌ ‌waiting‌ ‌for‌ ‌the‌ ‌centrifugation,‌ ‌label‌ ‌each‌ ‌cryovial.‌ ‌We‌ ‌recommend‌ ‌writing‌ ‌the‌ ‌name‌ ‌of‌ ‌the‌‌PSC‌‌line‌‌used,‌‌passage‌‌number,‌‌date,‌‌and‌‌the‌‌number‌‌of‌‌cells‌‌in‌‌the‌‌vial.‌ ‌4. Aspirate‌ ‌the‌ ‌supernatant‌ ‌as‌ ‌much‌ ‌as‌ ‌possible‌ ‌but‌ ‌not‌ ‌completely.The‌ ‌pellet‌ ‌should‌ ‌still‌‌be‌‌covered‌‌with‌‌a‌‌small‌‌volume‌‌of‌‌the‌‌supernatant.‌ ‌5. Tap‌‌the‌‌tube‌‌10‌‌times‌‌to‌‌loosen‌‌the‌‌pellet.‌ ‌6. Resuspend‌‌the‌‌pellet‌‌0.2‌‌ml‌‌/‌‌vial‌‌STEM‌‌CELLBANKER.‌ ‌7. Distribute‌‌0.2‌‌ml‌‌of‌‌the‌‌suspension‌‌to‌‌each‌‌cryovial.‌ ‌8. Make‌‌sure‌‌that‌‌the‌‌caps‌‌are‌‌closed‌‌tightly‌‌and‌‌transfer‌‌the‌‌cryovials‌‌into‌‌a‌‌Mr.‌‌Frosty‌‌Freezing‌‌Container.‌‌Make‌‌sure‌‌that‌‌Mr.‌‌Frosty‌‌contains‌‌250‌‌ml‌‌isopropanol.‌ ‌9. Loosely‌‌close‌‌the‌‌lid‌‌of‌‌Mr.‌‌Frosty‌‌with‌‌cryovials,‌‌put‌‌it‌‌into‌‌a‌‌-80°C‌‌freezer‌‌and‌‌leave‌‌it‌‌overnight‌‌or‌‌up‌‌to‌‌a‌‌few‌‌days.‌ ‌10. Transfer‌‌the‌‌cryovials‌‌into‌‌a‌‌liquid‌‌nitrogen‌‌storage‌‌tank.‌ ‌‌‌Appendix‌‌D‌ ‌Thawing‌‌cells‌‌ ‌Note:‌‌After‌‌thawing‌‌frozen‌‌cells,‌‌we‌‌recommend‌‌users‌‌culture‌‌cells‌‌in‌‌medium‌‌supplemented‌‌with‌‌ROCK‌‌inhibitor‌‌(Y27632)‌‌for‌‌48‌‌hours.‌ ‌‌1. Transfer‌‌10‌‌ml‌‌of‌‌iStemFit‌‌to‌‌a‌‌15‌‌ml‌‌conical‌‌tube‌‌and‌‌warm‌‌it‌‌in‌‌a‌‌37°C‌‌water‌‌bath‌‌for‌‌30‌‌minutes‌‌until‌‌it‌‌reaches‌‌
37°C.‌ ‌2. Get‌‌the‌‌cryovial‌‌of‌‌cells‌‌from‌‌LN2.‌‌ ‌3. Spray‌‌a‌‌Kimwipe‌‌with‌‌70%‌‌EtOH‌‌and‌‌wipe‌‌the‌‌cryovial.‌‌Transfer‌‌the‌‌cryovial‌‌to‌‌the‌‌hood.‌‌ ‌4. Add‌‌800‌‌μl‌‌of‌‌warm‌‌iStemFit‌‌to‌‌the‌‌vial‌‌using‌‌a‌‌P1000‌‌pipettor.‌ ‌5. Gently‌‌pipet‌‌up‌‌and‌‌down‌‌with‌‌the‌‌P1000‌‌until‌‌just‌‌thawed.‌‌ ‌6. Using‌ ‌the‌ ‌same‌ ‌tip,‌ ‌increase‌ ‌the‌ ‌P1000‌ ‌pipette‌ ‌volume‌ ‌setting‌ ‌from‌ ‌800‌ ‌μl‌ ‌to‌ ‌1000‌ ‌μl‌ ‌and‌ ‌transfer‌ ‌the‌ ‌cell‌‌suspension‌‌to‌‌a‌‌new‌‌15‌‌ml‌‌conical‌‌tube.‌ ‌7. Using‌‌a‌‌P1000‌‌pipettor,‌‌add‌‌1‌‌ml‌‌of‌‌warm‌‌iStemFit‌‌to‌‌the‌‌cryovial‌‌and‌‌pipet‌‌up‌‌and‌‌down,‌‌washing‌‌the‌‌sides‌‌of‌‌the‌‌vial,‌‌to‌‌obtain‌‌any‌‌cells‌‌remaining‌‌in‌‌the‌‌vial.‌‌ ‌8. Transfer‌‌the‌‌cell‌‌suspension‌‌to‌‌the‌‌same‌‌15‌‌ml‌‌conical‌‌tube‌‌with‌‌the‌‌same‌‌P1000‌‌pipette‌‌tip.‌‌ ‌9. Bring‌‌the‌‌volume‌‌in‌‌the‌‌15‌‌ml‌‌conical‌‌up‌‌to‌‌5‌‌ml‌‌by‌‌adding‌‌additional‌‌iStemFit‌‌using‌‌a‌‌5‌‌ml‌‌pipette.‌‌ ‌10. Centrifuge‌‌the‌‌cell‌‌suspension‌‌in‌‌the‌‌15‌‌ml‌‌tube‌‌at‌‌200‌‌x‌‌g‌‌for‌‌4‌‌minutes.‌ ‌11. Visually‌‌confirm‌‌that‌‌there‌‌is‌‌a‌‌pellet.‌‌Use‌‌a‌‌pasteur‌‌pipet‌‌to‌‌aspirate‌‌most‌‌of‌‌the‌‌supernatant‌‌from‌‌the‌‌conical‌‌tube,‌‌leaving‌‌a‌‌small‌‌volume‌‌of‌‌supernatant‌‌(<‌‌50‌‌μl)‌‌to‌‌cover‌‌the‌‌cell‌‌pellet.‌‌ ‌12. Tap‌‌the‌‌side‌‌of‌‌the‌‌conical‌‌tube‌‌up‌‌to‌‌10‌‌times‌‌to‌‌break‌‌up‌‌the‌‌cell‌‌pellet.‌‌ ‌13. Add‌ ‌1‌ ‌ml‌ ‌iStemFit‌ ‌to‌ ‌the‌ ‌conical‌ ‌tube‌ ‌using‌ ‌a‌ ‌P1000‌ ‌pipette‌ ‌and‌ ‌pipet‌ ‌up‌ ‌and‌ ‌down‌ ‌no‌‌more‌‌than‌‌2-3‌‌times‌‌to‌‌resuspend‌‌cells.‌ ‌14. Plate‌‌the‌‌cells‌‌as‌‌described‌‌in‌‌step‌‌13‌‌of‌‌Day‌‌0‌‌“Passaging”.‌‌
elixirgensci.com‌ ‌6‌‌