Note: To maintain sterile culture conditions, all of the procedures in this protocol are carried out using sterile laboratory practices and conducted under a laminar flow hood.
Prior to starting, ensure that any media is equilibrated to 37&deg;C and appropriately gassed.
Preparing Geltrex-coated Culture Dishes
Note: see appendix for the use of CELLstart coated culture dishes.
<ol>
 <li>Thaw one tube of Geltrex (1 mL) slowly at 2-8&deg;C and dilute 1:100 it in 99 mL of Knockout D-MEM/F-12. Mix the solution gently. 
 
 Note: Some iPS cell lines may require a different Geltrex dilution for optimal growth. See appendix for alternative dilutions.</li>
 <li>Cover the whole surface of each culture dish with the Geltrex solution (1 mL for a 35-mm dish, 1.5 mL for a 60-mm dish).</li>
 <li>Seal each dish with Parafilm to prevent drying and incubate the dishes for 1 hour at 37&deg;C. 
 
 Note: At this point you may store the Geltrex-coated culture dishes at 4&deg;C for up to 1 month. Seal each dish with Parafilm to prevent the Geltrex from drying out.</li>
 <li>Prior to using, transfer the Geltrex-coated dishes to a laminar flow hood and allow them to equilibrate to room temperature (about 1 hour).</li>
</ol>
Preparing Complete KnockOut SR Feeder-Free Medium
<ol>
 <li> To prepare 1 mL of 10 &mu;g/mL Basic FGF solution, aseptically mix the components listed below. Aliquot the solution and store at -20&deg;C for up to 6 months. 
 <table border="0">
 <tbody>
 <tr>
 <td>Basic FGF</td>
 <td>10 &mu;g</td>
 </tr>
 <tr>
 <td>D-PBS</td>
 <td>990&mu;L</td>
 </tr>
 <tr>
 <td>10% BSA</td>
 <td>10 &mu;L</td>
 </tr>
 </tbody>
 </table>
 
 Note: BSA can be substituted with HSA or Knockout SR at the same concentration.</li>
 <li>To prepare 50 mL of 2 mg/mL Dispase solution, aseptically mix the components listed below. Filter sterilize the solution and store at 4&deg;C for up to 2 weeks.
 <table border="0">
 <tbody>
 <tr>
 <td>Dispase</td>
 <td>100 mg</td>
 </tr>
 <tr>
 <td>D-PBS</td>
 <td>50 mL</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>To prepare 100 mL of complete KnockOut SR Feeder-Free (KSR-FF) aseptically combine the components listed in the table below. 
 <table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Stock Concentration</td>
 <td>Final Concentration</td>
 <td>Volume </td>
 </tr>
 <tr>
 <td>Knockout DMEM/F12 (Cat. no. 12660-012)</td>
 <td>-</td>
 <td>1X</td>
 <td>76.8 mL</td>
 </tr>
 <tr>
 <td>GlutaMAX -I (Cat. No. 35050-061)</td>
 <td>200 mM</td>
 <td>2 mM</td>
 <td>1 mL</td>
 </tr>
 <tr>
 <td>KnockOut SR (Cat. no. 10828-028)</td>
 <td>-</td>
 <td>20%</td>
 <td>20 mL</td>
 </tr>
 <tr>
 <td>KnockOut SR-GFC (Cat. no. A10580-01)</td>
 <td>50X</td>
 <td>1X</td>
 <td>2 mL</td>
 </tr>
 <tr>
 <td>bFGF (Cat. no. PHG0024).</td>
 <td>10 &mu;g/mL</td>
 <td>20 ng/mL</td>
 <td>200 &mu;L</td>
 </tr>
 </tbody>
 </table>
 
 You may store the KSR-FF medium at 2-8&deg;C for up to one week.</li>
 <li>Just before pre-equilibrating the complete medium to temperature and gases, aseptically add the required volume of 2 mercaptoethanol (55 mM stock concentration) for a 0.1 mM final concentration. For example, to prepare 100 mL of KSR-FF medium add 182 &mu;L of 55 mM 2-mercaptoethanol (1:550 dilution) Alternatively, the 2-mercaptoethanol may be added to the 1X completed medium and stored at 2-8&deg;C for up to one week.</li>
</ol>
Preparing Freezing / Cryopreservation Medium
The cryopreservation medium consists of two media; Freezing Medium A and Freezing Medium B. They are added to the cells at different times and must remain separated. They are each 50% of the total volume of Cryopreservation Medium needed. The total volume of Cryopreservation Medium needed is 1 mL for each vial to be frozen. A 90% confluent 60mm dish of hESC can be used to bank 2 vials of hESC in cryopreservation media.
Prepare enough volume of each Freezing Medium with an extra 2-5mL to ensure overage.
<table border="1">
 <tbody>
 <tr>
 <td>Freezing Medium A (50% of final volume):</td>
 <td>50% DMEM/F12</td>
 <td>50% KSR</td>
 </tr>
 <tr>
 <td>Freezing Medium B (50% of final volume):</td>
 <td>80% DMEM/F12</td>
 <td>20% DMSO</td>
 </tr>
 </tbody>
</table>
Example:
If you are freezing 20 vials of cells, you will need 20mL of Cryopreservation Medium. Add 4mL for overage for a total of 24mL Cryopreservation Medium. That means you will need 12mL of Freezing Medium A (50% of 24mL) and 12mL of Freezing Medium B (50% of 24mL).
<table border="1">
 <tbody>
 <tr>
 <td>Freezing Medium A (12 mL):</td>
 <td>6 mL DMEM/F12</td>
 <td>6 mL KSR</td>
 </tr>
 <tr>
 <td>Freezing Medium B (12 mL):</td>
 <td>9.6 mL DMEM/F12</td>
 <td>2.4 mL DMSO</td>
 </tr>
 </tbody>
</table>
The final composition of the Cryopreservation Medium is 65% DMEM/F12, 25% KSR, and 10% DMSO.
Cryopreserving iPSCs
<ol>
 <li>Pre-warm the required volume of Dispase in a 37 &deg;C water bath. Refer to Table 1 below for details on the volumes required. </li>
 <li>Pre-equilibrate the required volume of KSR-FF in a 37&deg;C water bath for15 min. Refer to Table 1below for details on the volumes required. </li>
 <li>Aspirate the spent medium from the culture vessel using a pipette, and rinse the cells twice with D-PBS.</li>
 <li>Gently add pre-warmed Dispase solution to the culture vessel (e.g., 1 mL of Dispase solution per 60-mm culture dish). Swirl the culture vessel to coat the entire cell surface.</li>
 <li>Incubate the culture vessel at 37&deg;C for 3 minutes.</li>
 <li>Remove the vessel from the incubator, aspirate the Dispase solution, and gently wash the cells with D-PBS.</li>
 <li>Gently scrape the cells off the surface of the culture dish using a cell scraper, and transfer the cells to a sterile 15mL centrifuge tube.</li>
 <li>Rinse the culture dish twice with KSR-FF, gently "spraying off" any cells that have not detached. Pool the rinse medium with the cells in the 15mL tube.</li>
 <li>Centrifuge the tube at 200 x g for 5 minutes at room temperature to pellet the cells.</li>
 <li>Carefully aspirate the supernatant without disturbing the cell pellet and discard it.</li>
 <li>Prepare a sufficient quantity of cryovials. Once the cells are in contact with DMSO, they should be aliquoted and frozen within 2-3 minutes.</li>
 <li>Gently flick the tube, to fully dislodge the cell pellet from the tube bottom and re-suspend the cells in Freezing Medium A. [by gently pipetting up and down using a 5-mL serological pipette]. Following uniform suspension of clumps, add equal volume of Freezing Medium B to it, in a drop wise manner, while gently swirling the cell suspension to mix. 
 
 Note: At this point, cells are in contact with DMSO, and work must be performed efficiently. Once cells are in contact with DMSO, they should be aliquoted and frozen within 2-3 minutes.</li>
 <li>Aliquot 1 ml of the cell suspension into each cryovial.</li>
 <li>Quickly place the vials in a Mr. Frosty [to rapidly freeze] and transfer it to -80&deg;C overnight.</li>
 <li>After overnight storage at -80&deg;C, transfer the cells to a liquid nitrogen tank for long term storage.</li>
</ol>
iPSC vial thaw and cell recovery
Note: KSR-FF may be replaced with KSR-containing MEF-conditioned medium, or KSR medium for use with feeders. See appendix for instructions for media preparation. 
Pre-warm 10mL of KSR-FF medium in a 50 mL tube.
<ol>
 <li>Equilibrate the appropriate quantity of Geltrex-coated dishes to room temperature and aspirate any liquid from the dishes just before plating your cells.</li>
 <li>Carefully remove the hESC (or iPSC) vial from Liquid Nitrogen Storage Tank.</li>
 <li>Rapidly thaw frozen vial of cells in a 37&deg;C water bath, until just a small frozen chunk remains in the vial.</li>
 <li>Spray vial with 70% isopropanol to decontaminate it and transfer it to the sterile tissue culture hood.</li>
 <li>Aseptically transfer the entire contents of the vial into a 15 mL conical tube using a 5 mL pipette.</li>
 <li>Rinse the vial with 1 mL of KSR-FF and add this to the 15 mL centrifuge tube.</li>
 <li>Slowly, add 4 mL of KSR-FF drop-wise to cells in the 15mL conical tube. While adding the medium, gently move the tube back and forth to mix the cells. (This reduces osmotic shock to the cells).</li>
 <li>Centrifuge the 15 mL tube with the cell suspension at 1000rpm (200 x g) for 2 minutes at room temperature.</li>
 <li>Carefully aspirate and discard the supernatant without disturbing the cell pellet.</li>
 <li>Re-suspend the cell pellet in pre-warmed KSR-FF (e.g. 4mL/60 mm dish) using a 5 mL pipette and gently pipette the cells up and down until cell pellet is fully dispersed. A viability of greater than 70% is expected, however if the viability is less than 70%, it may take longer for the cells to reach confluence.</li>
 <li>Using the same pipette, transfer the cell suspension drop-wise to Geltrex-coated culture dish pre-equilibrated to room temperature.</li>
 <li>Place the culture dish in a 37&deg;C incubator, with a humidified atmosphere of 4 to 6% CO2 in air. Carefully swirl vessel in a north to south, east to west pattern to evenly distribute the cells.</li>
 <li>Gently fluid-change the culture dish 24 hrs post-thaw and daily thereafter to remove cell debris and to provide fresh nutrients until the dish is approximately 70-80% confluent. For continued culture and passaging of your iPS cells, refer to our protocol titled "Feeder-Free Culture and Passaging of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium"</li>
</ol>
Expected Results
The cells should be roughly 70-80% confluent prior to cryopreservation. Dispase digestion results in curling up and folding of the colonies along the colony margins. After harvesting the cells they are frozen as small clumps in cryopreservation media. Once the vial is thawed, cells recover and attach to the pre-coated dish as small clumps. They grow rapidly leading to a confluent dish in 5-6 days.
Table 1 - Recommended Volumes
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>35mm Dish</td>
 <td>60mm Dish</td>
 <td>100mm Dish</td>
 </tr>
 <tr>
 <td>Complete KnockOut SR medium</td>
 <td>2 mL</td>
 <td>4 mL</td>
 <td>10 mL</td>
 </tr>
 <tr>
 <td>Geltrex Solution</td>
 <td>1 mL</td>
 <td>1.5 mL</td>
 <td>4-5 mL</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>0.5 mL</td>
 <td>1 mL</td>
 <td>3-4 mL</td>
 </tr>
 <tr>
 <td>D-PBS for rinsing</td>
 <td>2 mL</td>
 <td>4 mL</td>
 <td>10 mL</td>
 </tr>
 </tbody>
</table>
Please <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/2237/2237_appendix.pdf">click here to see the appendix</a>.

<ol>
	<li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

The authors of this article are employed by Life Technologies that produces reagents and instruments used in this article.

<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>12660-012</td><td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>17105-041</td><td>Note: see appendix for alternative passaging methods</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td><td></td><td>14190-144</td><td></td></tr><tr><td>DMSO, 500mL</td><td>JT Baker</td><td>JT9224-1</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60 mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen storage tank</td><td></td><td></td><td></td></tr><tr><td>Isopropanol freezing containers</td><td></td><td></td><td></td></tr><tr><td>1.5 mL Cryovials</td><td></td><td></td><td></td></tr></tbody></table>

cryopreserving and recovering of human ips cells using complete knockout serum replacement feeder-free medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

Universidad San Sebastian

Research

JoVE Journal

Biology

18.2K Views.  Life Technologies.  中文 日本語 

Video: Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

Developmental Biology

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Bioengineering

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...