Download User Manual - Cyagen Biosciences
Transcript
User Manual OriCellTM Sprague-Dawley (SD) Rat Mesenchymal Stem Cells with RFP (MSCs/RFP) Cat. No. RASMX-01201 Table of Contents Contents and Storage ……………………………………………………………………………………… 3 Product Introduction ……………………………………………………………………………………… 3 Cell Characteristics and Identity ……………………………………………………………………… 3 Product Application ………………………………………………………………………………………… 4 General Handling Principles …………………………………………………………………………… 4 Culturing OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP Thawing and Establishing OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP ……………… 4 Passaging Cyagen OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP ………………………… 6 Differentiation of OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP ……………………………… 7 Cryopreservation of OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP ……………………… 11 Appendix ……………………………………………………………………………….. 13 Troubleshooting ……………………………………………………………………………………………… 13 Related Products …………………………………………………………………………………………… 14 References …………………………………………………………………………………………………… 14 Technical Support ……………………………………………………………………. 15 CONTENTS AND STORAGE Product Name Sprague‐Dawley (SD) Rat Mesenchymal Stem Cells with RFP Catalog No. RASMX‐01201 Amount per Vial 1×106 Cells Cryopreserved At Fifth Passage Storage Condition Liquid Nitrogen CAUTION: Please handle this product as a potentially biohazardous material. This product contains dimethyl sulfoxide (DMSO), a hazardous material, in the freezing medium. PRODUCT INTRODUCTION Mesenchymal stem cells with RFP (MSCs/RFP) are multipotent stem cells that can differentiate into a variety of cell types including osteocytes, adipocytes, and chondrocytes. MSCs/RFP proliferate quickly and are capable of generating a local immunosuppressive microenvironment, thus contributing to their wide application potentials in tissue engineering, cell therapy, and gene therapy. OriCellTM Sprague-Dawley (SD) Rat Mesenchymal Stem Cells are derived from the bone marrow of Sprague-Dawley (SD) Rats, cultured as monolayer, and then have been transfected with a lentiviral construct containing a RFP expression motif. In addition, these cells have been tested for: • Exogenous Factors: bacterial/fungal contamination, mycoplasma contamination, and endotoxin contamination. • Characteristics: post-thaw viability, cell cycle, verification of undifferentiated state, and differentiation potential. This product is intended for laboratory research use only. It is not intended for diagnostic, therapeutic, clinical, household, or any other applications. CELL CHARACTERISTICS AND IDENTITY • Strong capacity to expand. Can be passaged at least 5 times. • Multipotent differentiation ability along the osteogenic, chondrogenic, and IMPI0074A2 RASMX‐01201 Page 3 of 15 adipo ogenic linea ages. • Posittive for CD2 29, CD44, a and CD90 (> ( 70%), and a negativ ve for CD34 4 and CD45 5 and CD11 1b (< 5%) in flow cytometry ass says. PRODUC CT APPL LICATIO ONS Sp prague-Daw wley (SD) Rat R MSCs/R RFP have be ecome a po opular resea arch targett due to th heir potential use in re egenerative e medicine and tissue engineering g (in areas such as ca ardiovascula ar, neural, and orthop pedic diseas se). P can be us sed as cell m models to evaluate e OriCellTM Sprrague-Dawlley (SD)Ratt MSCs/RFP he immunorreactions, proliferation p n, immigrattion, and differentiatio on of MSCs s/RFP both th in vivo and in n vitro. GENERA AL HAND DLING PRINCIP P PLES h of the producct is necess sary througho out. 1.. Aseptic handling 2.. Once the e cells have been estab blished, alw ways freeze e several vi als of OriCe ellTM Sprague--Dawley (SD)Rat MSC Cs/RFP as a backup. Note: The O OriCellTM Spr rague-Daw wley (SD) Ra at MSCs/RF FP can be ffrozen/thaw wed at leas st N on ne times. ecommende ed to use ce ells that are e at, or und der, an 3.. For all studies, it is strongly re p number of 10 0. original passage 4.. For general mainten nance of ce ells, we reco ommend th he seeding density to be 2.0cells/cm2. 3.0×104c 5.. For general mainten nance of ce ells, we reco ommend th hat the med dium is cha anged if it e pH indicattor in the medium m app pears yellow w). In general, becomes acidic (the very three days. d change the growth medium ev 6.. Do not le et OriCellTM M Sprague- Dawley (SD D)Rat MSCs s/RFP overg grow as it will w result in n contact in nhibition. When W the ce ells are 80--90% confluent, subcu ulturing the e cells is strongly recommend ded. Note: We sttrongly reco ommend th he use of Or riCellTM cult ture media and other related N re eagents for optimal re esults. THAWIN NG AND D ESTABLISHIN G OriCe ellTM SPRAGUE--DAWLE EY (SD) RAT MS SCs/RFP P M Materials Required R • OriCellTM Mesenchym mal Stem C Cell Growth Medium (C Cat. No. GU UXMX-9001 11) IMPI0074A2 RA ASMX‐01201 Page 4 of 15 Th hawing and a Estab blishing S prague-D Dawley(SD) Rat M MSCs/RFP P m the fully supplemen nted (complete) OriCellTM MSC Grrowth Mediium to 37°C C. 1.. Pre-warm 2.. Add 9 mL L of OriCellTM MSC Gro owth Mediu um to a 15 mL conical tube. 3.. Remove the cryovia al of OriCelllTM Sprague e-Dawley (S SD)Rat MSC Cs/RFP from m liquid nitrogen.. 4.. Quickly thaw t the cryovial in a 37°C water bath until the last icce crystal disappears. For optim mal results, be sure to o finish the thawing prrocedure wiithin 3 minutes. Be careful not to subm merge the en ntire vial. Maximum cell c viability y is depend dent on the d complete thawing off frozen cells. rapid and ess than op ptimal if the e cells are thawed t forr more than n 3 minutes s. N Note: Resultts will be le a the cells s are complletely thawed, disinfec ct the outsiide of the cryovial c 5.. As soon as with 70% % v/v ethan nol. TM 6.. Use a pip pette to transfer the ccells to the 15 mL coniical tube co ontaining OriCell O MSC Growth Medium m inside a biosafety cabinet. c Be e careful no ot to introdu uce any d the transfer prrocess. bubbles during 7.. Rinse the e vial with 1 mL of the e medium to t reduce cell loss. Su ubsequently y transfer this 1 mL L of cell sus spension in nto the conical tube. 8.. Gently mix m the cell suspension n by slowly pipetting up u and dow wn. Be care eful not to introduce e any bubbles. 9.. Centrifug ge the cell suspension s at 250 x g for 5 minu utes. 10 0. Carefully y aspirate off as much of the supernatant as s possible a and add 2-3 3 mL of fresh OriCellTM MSC Growth Me edium (pre-warmed to o 37°C). 11 1. Gently re esuspend th he cells in O OriCellTM MS SC Growth Medium. 12 2. Seed the e cells into a T25 flask k and add a sufficient amount a of OriCellTM MSC M Growth h Medium. Gently roc ck the cultu ure flask to o evenly dis stribute the e cells. 13 3. Incubate e the flask at a 37°C ins ide a 5% CO C 2 humidiffied incubattor. 14 4. The nextt day, chang ge the med dium with fresh f growth medium (pre-warmed to 37°C C). 15 5. Change the t growth medium ev very three days thereafter. 16 6. When the e cells are approximattely 80-90% % confluent, they can n be dissociated with Trypsin-E EDTA and passaged. p Note: Chang ging Mediium N n appropriatte amount of medium to 37°C in a sterile co ontainer. Replace R the e 1.. Warm an spent me edium with the pre-wa armed, fres sh medium.. Once com mpleted, retturn the flask to the t incubator. 2.. Avoid rep peated war rming and c cooling of the medium m. If the en ntire conten nt is not needed fo or a single procedure,, transfer only o the req quired volu me to a ste erile secondarry container. IMPI0074A2 RA ASMX‐01201 Page 5 of 15 Fig. 1. OriCellTM Sprague-Dawley (SD) Rat Mesenchymal Stem Cells with RFP are established. PASSAGING OriCellTM SPRAGUE-DAWLEY (SD)RAT MSCs/RFP Materials Required • 0.25%Trypsin-0.05%EDTA (Cat. No. TEDTA-10001) • Phosphate-Buffered Saline (1×PBS) (Cat. No. PBS-10001) • OriCellTM Sprague-Dawley (SD) Rat Mesenchymal Stem Cells With RFP (Cat. No. RASMX-01201) • OriCellTM Mesenchymal Stem Cell Growth Medium (Cat. No. GUXMX-90011) Passaging OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP 1. Pre-warm the OriCellTM MSC Growth Medium, 1×PBS, and 0.25%Trypsin-0.05%EDTA solution to 37°C. 2. Carefully aspirate the spent medium from the 80-90% confluent monolayer of MSCs/RFP. 3. Add 1×PBS (6 mL for T75 flask, 3 mL for T25 flask). Be careful not to disturb the monolayer. Gently rock the flask back and forth to rinse the monolayer. 4. Aspirate 1×PBS off and discard. 5. Repeat steps 3-4 two or three times. 6. Add 0.25%Trypsin-0.05%EDTA solution (2-3 mL for T75 flask, 1 mL for T25 flask). Gently rock the flask back and forth to ensure that the entire monolayer is covered with the Trypsin-EDTA solution. Allow trypsinization to continue until the majority of the cells (approximately 80%) are rounded up. At this point, gently tap the side of the flask to release the majority of cells from the culture flask surface. Important: Avoid leaving cells exposed to the trypsin longer than necessary (no more than two minutes if using Cyagen’s trypsin-EDTA solution). Care should also be taken that the cells are not forced to detach prematurely as this may result in clumping. 7. After the cells are visibly detached, immediately add the pre-warmed OriCellTM MSC IMPI0074A2 RASMX‐01201 Page 6 of 15 Growth Medium M (6 mL for T75 5 flask, 3 mL for T25 flask) to neu utralize the e trypsiniza ation. 8.. Gently piipette the medium m ove er the cells to dislodge e and resusspend the cells. c Repeat 5-6 times un ntil all the ccells are dis ssociated frrom the fla ask and eve enly d into a single cell susspension. dispersed 9.. Transfer the dissocia ated cells in nto a 15 mL m conical tu ube. 10 0. Centrifug ge at 250 x g for 5 min nutes. 11 1. Carefully aspirate off as much of the supe ernatant as s possible. T MSC Gro 12 2. Add 2 mL L of OriCellTM owth Mediu um to the co onical tube e and gently y resuspend d the cells thoroughly y. 13 3. Plate the cells into appropriate a e flasks. OriCellTM Sprrague-Dawlley (SD) Ra at FP can be sp plit at 1:2 o or other appropriate ratios. MSCs/RF 14 4. Add an appropriate amount off medium to o the cells. Incubate tthe cells at 37°C inside e O2 humidifie ed incubato or. a 5% CO N Note: Care should be taken t to av void introdu ucing bubbles during pipetting. Additional Tips Tiime to Cha ange Mediium It is recomm mended to change c the culture me edium if the ere are too many dead d cells afterr pa assaging. It is recomm mended to change c the culture me edium when never the m medium bec comes ac cidic, even if the cells do not rea ch 80-90% % confluency y. The pH iindicator in the culture e m medium will appear yellow when a acidic. bculture Tiime to Sub W When OriCelllTM Sprague e-Dawley(S SD) Rat MS SCs/RFP are e 80-90% confluent, it is re ecommende ed that the e cells be su ubcultured.. Do not le et the cells o overgrow as a it will re esult in contact inhibition. OriCellTTM SPRAG GUE-DA AWLEY ( SD) RAT T MSC DIFFERE D ENTIATI ION T TM USING OriCell DIFFE ERENTIA ATION MEDIA M P can differrentiate into o a variety of cell OriCellTM Sprrague-Dawlley(SD) Ratt MSCs/RFP ypes including osteocy ytes, adipoccytes, and chondrocyt c tes. ty O Osteogenic Differen ntiation M Materials Required: R senchymal Stem Cell O Osteogenic c Differentia ation Mediu um (Cat. No o. GUXMXOriCellTM Mes 0021) 90 IMPI0074A2 RA ASMX‐01201 Page 7 of 15 sis Protoco ol Osteogenes Note: The protocol listed below iss for 6-welll tissue cultture platess. N M Sprague--Dawley (SD D) Rat MSC Cs/RFP in O OriCellTM Me esenchymal 1.. Culture the OriCellTM M at 3 37°C in a 5% CO2 hum midified inccubator. Stem Celll Growth Medium 2.. When cellls are apprroximately 80-90% co onfluent, they can be d d with dissociated 0.25%Try ypsin-0.04% %EDTA (Ca at. No. TED DTA-10001). 3.. Reseed the MSCs/R RFP in the g rowth med dium at 3×1 104 cells/cm m2 in a 6-w well tissue oated with 0 0.1% gelatin solution. culture plate pre-co 4.. Incubate the cells at 37°C insi de a 5% CO2 humidifiied incubattor. 5.. When cellls are apprroximately 60-70% co onfluent, ca arefully asp pirate off the growth TM medium from each well and ad dd 2 mL of OriCell Mesenchyma M ell al Stem Ce nic Differentiation Med dium. Osteogen 6.. Feed cells every thrree days forr 2-4 weeks by completely replaccing the medium with h CellTM Mese enchymal S Stem Cell Osteogenic O Differentiat D tion Medium m (prefresh OriC warmed to t 37°C). 7.. After 2-4 4 weeks of differentiat d ion, cells ca an be fixed and staine ed with aliz zarin red S. event osteoblasts from m detaching g, it is recommended tto change half h of the ote: To pre No me edium everry two days s before ana alysis. d S Stainin ng Analysiis Allizarin Red e cells have differentia ated, remov ve the osteo ogenic diffe erentiation medium 1.. After the from the wells and rinse with 1x phospha ate-buffered saline (PB BS). Fix ce ells with 2 mL of 4% % formaldehyde solutiion for 30 minutes. m 2.. Rinse we ells twice with 1x PBS.. Stain the cells with 1 mL alizarrin red S working solution for f 3-5 min nutes. 3.. Rinse we ells 2-3 time es with 1x PBS. 4.. Cells can n now be vis sualized an nd analyzed d under a microscope. m IMPI0074A2 RA ASMX‐01201 Page 8 of 15 Fig. 3 OriCellTM Sprague-Dawl S ley(SD) Rat MSCs/RFP M are differentiated into osteocyttes and a are stained with alizarin red d S. Adipogenic Differen ntiation M Materials Required R : OriCellTM Mes senchymal Stem Cell A Adipogenic c Differentia ation Mediu um (Cat. No o. GUXMX0031) 90 Ad dipogenes sis Protoco ol Note: The protocol listed below iss for 6-welll tissue cultture platess. N M 1.. Culture the t OriCellTM Sprague--Dawley (SD) Rat MSC Cs/RFP in th he OriCellTM Mesenchymal Stem Cell Growtth Medium at 37°C in a 5% CO2 humidified d incubator. 2.. When cellls are apprroximately 80-90% co onfluent, they can be d dissociated d with 0.25%Try ypsin-0.04%EDTA (Ca at. No. TED DTA-1000). 3.. Reseed the MSCs in n growth me edium at 2x104 cells/c cm2 in a 6--well tissue culture h a medium m volume o of 2 mL per well. plate with 4.. Incubate the cells at 37°C in a 5% CO2 humidified h incubator. 5.. Feed the cells every y three day ys until they y are 100% % confluent or post-confluent. Induction n of adipoge enic differe entiation at post-confluency is strrongly reco ommended.. 6.. When the e cells are 100% conffluent or po ost-confluen nt, carefully y aspirate off o the spent gro owth mediu um from the e wells and d add 2 mL of OriCellTMM Mesenchy ymal Stem Cell Adipogenic Differentiation medium A (induction medium) per well. 7.. Three days later, ch hange the m medium to OriCellTM Mesenchyma M al Stem Cell nic Differentiation med dium B (ma aintenance medium) b by complete ely Adipogen replacing g the spent medium A . 8.. 24 hours later, chan nge the me edium back to MSC Ad dipogenic D Differentiatio on medium m A. 9.. To optimally differentiate MSC Cs into adipo ogenic cells s, repeat th he cycle of induction IMPI0074A2 RA ASMX‐01201 Page 9 of 15 and maintenance at least three times. 10. After three to five cycles of induction and maintenance, culture the cells in OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation medium B for an additional 4-7 days until the lipid droplets are big, round enough. During these days period, change the medium every three days. Oil Red O Stain Analysis 1. After the cells have differentiated, remove the MSC maintenance medium from the wells and rinse with 1x phosphate-buffered saline (PBS). Fix cells with 2 mL of 4% formaldehyde solution for 30 minutes. 2. Rinse wells twice with 1x PBS and stain cells with 1 mL of oil red O working solution (3:2 dilution with distilled water and filter with filter paper) for 30 minutes. 3. Rinse wells 2-3 times with 1x PBS. 4. Cells can now be visualized and analyzed under a microscope. TM Fig.4 OriCell Sprague-Dawley(SD) Rat MSCs/RFP are differentiated into adipocytes and are stained with oil red O. Chondrogenic Differentiation Materials Required OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Cat. No. GUXMX-90041) Chondrogenesis Protocol 1. Calculate the total number of MSC pellet cultures required for your experiment (2.5×105 MSCs/RFP are needed to form each chondrogenic pellet). Transfer this amount of cells into an appropriate culture tube. 2. Wash the MSCs/RFP with Incomplete Chondrogenic Medium. Centrifuge the cells at 150 x g for 5 minutes at room temperature and then aspirate off the supernatant. IMPI0074A2 RASMX‐01201 Page 10 of 15 Resuspend the cells in 1 mL of Incomplete Chondrogenic Medium per 7.5×105 cells. Centrifuge again at 150 x g for 5 minutes and then aspirate off the medium. 3. Resuspend the MSCs/RFP in Complete Chondrogenic medium to a concentration of 5.0×105 cells/mL. 4. Aliquot 0.5 mL (2.5×105 cells) of the cell suspension into 15 mL polypropylene culture tubes. Centrifuge the cells at 150 x g for 5 minutes at room temperature. DO NOT aspirate the supernatant or resuspend the pellet. 5. Loosen the caps of the tubes one half turn in order to allow gas exchange, and incubate the tubes at 37°C in a humidified atmosphere of 5% CO2. Do not disturb the pellets for 24 hours. 6. Feed the cell pellets every 2-3 days by completely replacing the medium in each tube (to avoid aspirating the pellets when aspirating the medium, attach a sterile 1200μL pipette tip to the end of the aspirating pipette). Add 0.5 mL of freshly prepared Complete Chondrogenic Medium to each tube. 7. After replacing the medium, flick the bottom of the tube to ensure that the pellet is free floating. Loosen the caps and return the tubes to the 37°C incubator. 8. Chondrogenic pellets should be harvested after 14-28 days in culture. Pellets may be formalin-fixed and paraffin-embedded for alcian blue stain analysis. Alcian Blue Staining Procedure 1. The tissue sample should be formalin-fixed and paraffin-embedded already. 2. Staining procedure: a) Deparaffinize slides and hydrate to distilled water. b) Stain in alcian blue solution for 30 minutes. c) Wash in running tap water for 2 minutes. d) Rinse in distilled water. e) Visualize under a light microscope and capture images for analysis. Blue staining indicates synthesis of proteoglycans by chondrocytes. Fig.5 OriCellTM Sprague-Dawley (SD) Rat MSCs/RFP are differentiated into cartilages and are stained with alcian blue. IMPI0074A2 RASMX‐01201 Page 11 of 15 CRYOPR RESERVA ATION OF O CELL LS USIN NG OriCe ellTM CRYOPR RESERVA ATION MEDIA M R Protein-F Free Cryoprreservation Medium (C Cat. No. NC CPF-10001) is a OriCellTM NCR use freezing g medium. Its chemically-define ed and prottein-free prrotein-free,, ready-to-u fo ormulation has h been optimized to o stem cells s and prima ary cells, th hus greatly enhancing th he viability and integrity of these cells by prrotecting th hem from da amage durring the on ne-step free eze-thaw procedure. p Unlike other conventiional freezi ng media, which re equire a slow programmed freeze e, this prod duct allows the cells to o be directly y frozen at 80 0°C. Cryopreservation ge the cultu ure medium m with fresh h growth medium m 24 h hours before freezing Note: Chang g. N ells that are e in the log garithmic growth phas se. Perform m a cell count to 1.. Collect ce determin ne the viable cell dens ity. 2.. Centrifug ge the cells for 3-5 mi nutes at 25 50 x g and 20°C. Rem move and discard the supernattant using a pipette. 3.. Resuspen nd the cell pellet in th e OriCellTM NCR Protein-Free Cry yopreservation Medium m at a cell density d of 10 1 5-106 cellls/mL. 4.. Dispense e aliquots of the cell su uspension into cryogenic storage e vials that are properly labeled. 5.. Place the e vials direc ctly in a -80 0°C freezerr. After 24 hours, tran nsfer the fro ozen vials to liquid nitrogen fo or long-term m preservattion. IMPI0074A2 RA ASMX‐01201 Page 12 of 15 APPENDIX Troubleshooting The table below lists some potential problems and solutions for culturing MSCs/RFP. Problem Low cell recovery rate Cause Solution The storage condition does not meet the requirements Purchase a replacement and store in liquid nitrogen for long‐term preservation. Thawing of the cells takes too long Thaw cells for no more than 3 minutes. Cells are incompletely recovered after thawing After aspirating off medium, wash the tube with culture medium twice and transfer all of the cells to the dish. Cells are handled roughly Care should be taken to avoid introducing bubbles during pipetting. Also avoid vortexing and high‐speed centrifugation Medium is not pre‐warmed Warm medium to 37°C before recovery. Mycoplasma contamination Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells. Slow cell growth Over digestion Wash the cells with PBS 2‐3 times to remove serum prior to trypsinization (serum will inhibit the function of trypsin). Control the digestion time. Cell aging Plating density is too low Increase the plating density. Inappropriate serum and medium Use Cyagen tailor‐made culture media. If other serum and media products are used, please perform validation to ensure compatibility. Dead cells are not removed promptly Change the medium next day after recovery to ensure removal of all dead cells. Cell Contamination Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells. Plating density is too low Some stem cells can secrete factors to support cell growth. Therefore, a certain degree of plating density must be maintained; otherwise, it will lead to cell proliferation slow down and cell aging. IMPI0074A2 RASMX‐01201 Page 13 of 15 Over digestion Cell aging Wash the cells with PBS 2‐3 times to remove serum prior to trypsinization (serum will inhibit the function of trypsin). Cells show spontaneous differentiation Ineffective induction of cell differentiation Control the digestion time. The passaging time is not appropriate The cells should be subcultured when reaching 80‐90% confluency in order to avoid contact inhibition. DMSO is not completely removed during cell recovery Wash the cells with pre‐warmed medium 2‐3 times during recovery. Differentiation reagents need to be optimized Cell passage is too high Use Cyagen tailor‐made differentiation media. Use cells at a low original passage number. RELATED PRODUCTS Product Catalog Number OriCellTM Mesenchymal Stem Cell Growth Medium GUXMX-90011 OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium GUXMX-90021 OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Medium GUXMX-90031 OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium GUXMX-90041 0.25%Trypsin-0.04%EDTA TEDTA-10001 Phosphate-Buffered Saline (1xPBS) PBS-10001 OriCellTM NCR Protein-Free Cryopreservation Medium NCPF-10001 REFENRENCES Jiang, Yuehua, Jahagirdar, Balkrishna N, and Reinhardt, R Lee.(2002)Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41-49. Hideya Yoshimura, Takeshi Muneta, and Akimoto Nimura. (2006)Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell and Tissue Research 327:449-462. IMPI0074A2 RASMX‐01201 Page 14 of 15 Cyagen Biosciences reserves all rights on the technical documents of its OriCellTM cell culture products. No part of this document may be reproduced or adapted for other purposes without written permission from Cyagen Biosciences. IMPI0074A2 RASMX‐01201 Page 15 of 15