Name: Culture and Maintenance of Human Embryonic Stem Cells (Video) | JoVE Journal
Uploaded: 2009-12-22T16:50:00.000+00:00
Duration: 9 min 36 s
Description: Learn to maintain healthy, undifferentiated human embryonic stem cells using daily feeding, manual differentiation removal, and collagenase passaging techniques. This protocol ensures optimal cell viability for research applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

1. Maintenance: Observing Cultures Under the Microscope
 <ol>
 <li>Remove one plate of hES cells from the 37°C incubator and bring to the microscope.</li>
 <li>Observe the colonies at low power, using 2X or 5X magnification, to assess the overall culture quality. Note the following: normal medium color, absence of any visible contamination, overall colony size, quality and density, and any other observations.</li>
 <li>Observe medium color changes. Due to the change in pH and the depletion of nutrients in the medium after an overnight incubation with the hES cells, the medium will change from a red hue to a "straw" color. If the medium appears purple, a basic pH shift has occurred. If the medium appears extremely yellow, the medium has acidified. Very yellow medium may be the result of extremely overcrowded colonies in the well or contamination. The medium should appear clear at all times. Though a certain level of cell debris in the medium is normal, it should never appear cloudy. If a high level of cell debris is observed, the culture is not healthy and may be contaminated.</li>
 <li>If the cultures do not require maintenance or passaging, they can be taken to the biological safety cabinet to be fed (see section 2).</li>
 <li>If the cultures contain more than approximately 10% differentiating colonies, the cells should be "cleaned up" by manually removing the differentiated areas (see section 3). </li>
 <li>If the cultures contain large or dense colonies, or the MEF feeder layer is more than 12 days old, the cells should be passaged (see section 4).</li>
 </ol>
2. Feeding hES Cells
 <ol>
 <li>In a sterile biological safety cabinet, remove the lid of the culture dish and aspirate the spent medium. Do not allow the tip of the pipette to touch any part of the plate other than directly inside the wells. If there is any concern of contamination, change to a new, sterile pipette.</li>
 <li>Using a sterile glass pipet, add the appropriate amount of warmed hES cell culture medium to each well. For cultures in a 6-well plate, add 2.5 mL medium per well.</li>
 <li>Return the plate to the incubator to remain at 37°C and 5% CO2.</li>
 </ol>
3. Removing Differentiation from hES Cell Cultures
 <ol>
 <li>Transform 9 inch glass Pasteur pipettes into picking tools by molding the tips over the controlled flame of an alcohol burner. Be sure that the tip of the pipette is sealed to prevent contamination and rounded to avoid scratching the plastic of the culture dish. Sterilize the picking tools before use using the UV light source in the biological safety cabinet or picking enclosure.</li>
 <li>Remove the differentiated areas. Differentiation often occurs in only a portion of an hES cell colony, usually along the edges or as isolated spots in the center. If only a section of a colony is differentiating, only the differentiated area needs to be removed. Differentiation can often lift off the plate in a "sticky" sheet, and it is helpful to first separate the differentiated cells from the rest of the colony by drawing a line through the colony with the end of the picking tool.</li>
 <li>Once the pieces of the colony are separated, gently glide the picking tool along the plate to detach the unwanted cells. Take care not to scrape away too much of the MEF feeder layer between the colonies in this process.</li>
 <li>When all of the differentiated cells are removed from the plate (and floating in the medium), return the culture to the biological safety cabinet. Aspirate the medium containing the differentiated cells and replace with fresh hES cell culture medium.</li>
</ol>
4. Passaging hES Cells
Enzyme Incubation
 <ol>
 <li>In a sterile biological safety cabinet, remove the lid of the 6-well culture dish and aspirate the spent medium from the wells to be passaged. Do not allow the tip of the pipette to touch any part of the plate other than directly inside the wells. </li>
 <li>Using a sterile glass pipet, add 1 mL of warmed collagenase IV to each well to be passaged.</li>
 <li>Return the plate to the 37° C incubator for 5 minutes.</li>
 </ol>
Scraping and Pooling the hES Cells
 <ol start="4">
 <li>After incubating the cells with collagenase, observe the colonies under the microscope. The enzyme should cause a subtle but observable change in the colony edges.</li>
 <li>In the biological safety cabinet, gently aspirate the enzyme from each well and replace with 2.0 mL hES cell culture medium.</li>
 <li>Tip the culture plate slightly toward you and take up the 2.0 mL medium in the first well with a 5 mL glass pipet. Holding the pipet perpendicular to the bottom of the plate, gently glide the pipet tip across the well while slowly releasing the medium.</li>
 <li>Repeat the scraping and pipetting motion 3-4 times until all of the colonies have been removed from the well. Pipet gently to avoid breaking up the colonies into too small of pieces. When the cells have been removed from the first well, leave the contents in the well and begin scraping the cells off of the next well.</li>
 <li>When all of the wells to be passaged have been scraped, pool the medium containing the colony pieces from each well in a sterile 15 mL conical tube.</li>
 <li>Rinse the scraped wells by adding 1 mL of hES cell culture medium to each well. Collect this rinse and transfer to the conical tube.</li>
 <li>Pipet the cells gently in the tube to break up the colony pieces up to the desired size.</li>
 <li>Centrifuge the cells for 5 minutes at 200 x g.</li>
 </ol>
Plating the hES Cell Cells
 <ol start="12">
 <li>While the hES cells are in the centrifuge, prepare a new 6-well MEF feeder plate. Label the plate with the appropriate hES cell information: cell line, new passage number, and passage date. Aspirate the MEF medium from the wells and add 1.0 mL PBS to each well. Gently swirl the buffer around the wells and aspirate the PBS wash. Add 1.5 mL hES cell culture medium to each well and set the plate aside.</li>
 <li>When the hES cells are finished centrifuging, bring the tube back to the biological safety cabinet. Aspirate the supernatant, being careful not to disturb the loosely-packed cell pellet.</li>
 <li>Resuspend the cells in the pellet with enough medium for 1 mL hES cell culture medium per well to be plated. When splitting into 6 new wells, resuspend the pellet in 6 mL medium.</li>
 <li>Gently and evenly add 1 mL of the cell suspension to each prepared well of the MEF feeder plate.</li>
 <li>Place the plate into the 37°C incubator and carefully slide the plate forward to back, and side to side to evenly distribute the cells throughout the well.</li>
 <li>Allow the cells to attach at 37°C overnight.</li>
 </ol>
5. Representative Results:
Undifferentiated hES cells are small, tightly-packed, and usually consist of larger, more spread out cells. Differentiation can occur within a colony (Figure 1A) or between colonies (Figure 3B).
Different morhopologies can be seen under low magnification under the microscope. A good cell morphology, as seen in Figure 2A, contains small, tightly packed cells that grow in a monolayer. Cells should have clean, defined edges, with little to no differentiation. The cells shown in Figure 2B are ready for passaging, and the cells shown in the Figure 2C are overcrowded.
<img src="/files/ftp_upload/1427/1427fig1.jpg" alt="Differentiation process, H1 cells, 10x microscopy images, colony formation analysis." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Differentiation in hES cultures. (A) differentiation of a colony (B)differentiation between colonies.
<img src="/files/ftp_upload/1427/1427fig2.jpg" alt="Cell culture morphology; microscope images at 10x magnification showing colony conditions." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. Morphologies seen in hES cultures. (A)good cell morphology (B) hES cells that are ready for passaging (C) overcrowded cells.

<ol><li>Freshney, R. I. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFreshney%2C+RI+%22Culture+of+Animal+Cells%3A+A+Manual+of+Basic+Technique%22">Culture of Animal Cells: A Manual of Basic Technique</a>. , 5th edition, Wiley-Liss. (2005).</li>
</ol>

Sterility must be maintained at all times when working with hES cells. Clean and sterilize the biological safety cabinet and all equipment before use. All reagents must be filtered using a 0.22 &#956;m pore size filter prior to use. The use of antibiotics in hES cell culture is not necessary and should be avoided.
A separate environment should be set up as a designated picking station. The sterile enclosure for the station can be a static enclosure (such as a PCR workstation) with a UV light source, a laminar flow hood, or a biological safety cabinet. A dissecting microscope inside the picking station is needed to observe the colonies as the differentiated areas are removed. The front glass panel of a static enclosure or a biological safety cabinet must be modified to allow for the oculars of the microscope to extend through the panel without compromising proper airflow and sterility.
To maintain healthy hES cell cultures, cells must be passaged at the optimal time, typically every 4-7 days. At this time the colonies have reached their maximum size and may be beginning to merge. Merged colonies can increase the rate of differentiation in the culture. Split ratios generally fall between 1:3 and 1:5, depending on the number of colonies plated, expansion rate, and culture conditions. Overplated colonies (split ratio too low) will likely merge prematurely and need to be passaged before they reach their maximum size.
Cultures on an MEF feeder layer that is more than 2 weeks old must be passaged on to new MEFs that will support undifferentiated growth and proliferation.
Since differentiation naturally occurs in hES cell cultures, a small amount is expected. Frequent or excessive differentiation may occur if the cultures do not receive appropriate care. The cells must be fed every day, overgrowth between passages should be avoided. Always use fresh reagents. All of the media, MEFs and reagents should be used within 14 days to avoid undifferentiated hES cell growth. New lots of reagents, such as Knockout Serum Replacement, FBS and MEFs, should be screened prior to use. Remember that any differentiated cells not removed prior to passaging will be transferred to the new cultures. Also, try to keep the cells at 37&#176;C whenever possible. Minimize the time the cells spend outside of the incubator (e.g. on the microscope stage or in the biological safety cabinet.) A heated microscope stage can be very beneficial if the cells will be of the incubator for extended periods of time.
When observing the hES cell cultures under the microscope, first assess the overall quality and size of the colonies view using a low power (2x or 5x) objective. If potentially differentiated cells are observed at low power, confirm the differentiated morphology using a higher magnification objective (10X).
Immediately after removing differentiation, the edges of the colonies may appear jagged or damaged. Incubate the colonies overnight in fresh medium to allow the remaining undifferentiated cells to recover. Without the influence of differentiation in the culture, these remaining colonies will continue to proliferate normally.
If possible, begin experiments using low-passage hES cells. Karyotype the cells before and after all major experiments.

<table><tbody><tr><td>D-MEM</td><td>Invitrogen</td><td>11965-092</td><td>For MEF medium</td></tr><tr><td>Fetal Bovine Serum (FBS), Certified</td><td>Invitrogen</td><td>16000-044</td><td>Heat-inactivated&lt;br/&gt;For MEF medium</td></tr><tr><td>D-MEM/F-12</td><td>Invitrogen</td><td>11330-057</td><td></td></tr><tr><td>Knockout&amp;#8482; Serum Replacement</td><td>Invitrogen</td><td>10828-028</td><td>Pre-screen to ensure support of hES cells</td></tr><tr><td>bFGF</td><td>Stemgent</td><td>03-0002</td><td>Use at [4 ng/mL] final</td></tr><tr><td>Non-essential Amino Acids</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>25030-081</td><td>200 mM (100X)&lt;br/&gt; Use at [1X] final</td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190-250</td><td>Without Ca&lt;sup&gt;2+&lt;/sup&gt; or Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Collagenase IV</td><td>Invitrogen</td><td>17104-019</td><td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td>Type A, Porcine</td></tr><tr><td>Fetal Bovine Serum (FBS), Defined</td><td>Hyclone</td><td>SH300.70.01</td><td>For freezing medium</td></tr><tr><td>6-well plates</td><td>Nalge Nunc international</td><td>140675</td><td>For general culture</td></tr><tr><td>4-well plates</td><td>Nalge Nunc international</td><td>176740</td><td>For thawing</td></tr><tr><td>5 mL glass pipets</td><td>Fisher Scientific</td><td>13-678-27E</td><td>Individually wrapped</td></tr><tr><td>15 mL conical tubes</td><td>Corning</td><td>430052</td><td>Polypropylene</td></tr><tr><td>Pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td>9&amp;#8221; glass</td></tr></tbody></table>

culture and maintenance of human embryonic stem cells  

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Hi, I'm Leah Kent from STEM Gent. Today we'll show you procedures for feeding, removing differentiation from and packaging human embryonic stem cells. We use these techniques in our lab for routine culture and maintenance of HES cells.So let's get started. The overall culture quality is assessed by observing the colonies at low power using two x or five x magnification. As you look at the cells under the microscope, note the following normal medium color absence of any visible contamination, colony size and density, and the overall quality of the cells.The fresh human ES cell culture medium normally has an orange or straw color after overnight incubation. The culture medium often appears more yellow due to the change in pH and the depletion of nutrients in the medium. If the medium appears purple as shown in this example, a basic pH shift has occurred.If the medium appears extremely yellow, the medium has acidified, which may be the result of extremely overcrowded colonies or contamination. The medium should appear clear at all times. If the culture does not contain large or dense colonies, the cells do not need packaging but do need daily feeding.Take the cells to the biological safety cabinet. Remove the lid of the culture dish and aspirate the spent medium. Do not allow the tip of the pipette to touch any part of the plate other than directly inside the wells.Using a sterile glass pipette at the appropriate amount of warm human ES cell culture, medium to each well for cultures in a six well plate at 2.5 milliliters of medium per well after the cells have been fed, return the plate to the incubator. Differentiation commonly occurs at the edges of human embryonic stem cell colonies. Using a microscope and a sterile picking tool, the differentiated cells can be gently removed from the edge of the colony, leaving behind the undifferentiated cells.For continued culture, the same procedure can be used when removing differentiated cells from the center of a colony, leaving a ring of undifferentiated human ES cells around the area of cells that were removed. If the culture contains more than approximately 10%differentiating colonies, the cells should be cleaned up by manually removing the differentiated areas. To do this first, transform nine inch glass past your pipettes into picking tools.By molding the tips over the controlled flame of an alcohol burner, be sure that the tip of the pipette is sealed to prevent contamination and rounded. To avoid scratching the plastic of the culture dish before using the picking tools, sterilize them using the UV light source in the biological safety cabinet for 15 to 20 minutes. To remove differentiated cells, first separate them from the rest of the colony by drawing a line through the colony with the end of the picking tool.Once the pieces of the colony are separated, gently glide the picking tool along the plate to detach the unwanted cells. Take care not to scrape away too much of the meth feeder layer between the colonies in this process. When all of the differentiated cells are removed from the plate and floating in the medium, take the culture to the biological safety cabinet.Aspirate the medium containing the differentiated cells and replace with fresh human ESL culture. Medium to passage. Human E es cells.The colonies are gently scraped from the surface of the culture plate using a serological pipette after a short enzyme incubation wants all of the wells to be passaged are scraped. The colony pieces are collected in a 15 milliliter conical tube and centrifuge to perform a loosely packed cell pellet. The supernatant is then removed and the cell pellet is resuspended to appropriately sized cell.Aggregates by pipetting in fresh human ESL medium. After rinsing a new culture plate containing a freshly irradiated meth feeder layer, the human E es cells are evenly distributed among the wells of the new plate and allowed to attach overnight. If the culture contains large or dense colonies, or the MEF feeder layer is more than 12 days old, the cells should be passaged in a sterile biological safety cabinet.Remove the lid of the six well culture dish and aspirate the spent medium from the wells to be passaged. Using a sterile glass pipette, add one milliliter of warmed collagenase four to each well to be passaged. Return the plate to the 37 degrees Celsius incubator for five minutes.After the five minutes, observe the colonies under the microscope. The enzyme should cause a subtle but observable change in the colony edges. They become more defined and a light halo may appear around them.In the biological safety cabinet, gently aspirate the enzyme from each well and replace with two milliliters of human ES cell culture.Medium. Tip the culture plate slightly toward you and take up the two milliliters of medium. In the first well with a five milliliter glass pipette, holding the pipette perpendicular to the bottom of the plate gently glide the pipette tip across the well.While slowly releasing the medium. Repeat this scraping and pipetting motion three to four times until all of the colonies have been removed from the well. When the cells have been removed from the first, well leave the contents the well and begin scraping the cells off of the next well.When all of the wells to be passaged have been scraped, pool the medium containing the colony pieces from each well in a sterile 15 milliliter conical tube pipette gently to avoid breaking up the colonies into pieces that are too small. Small rinse the scraped wells by adding one milliliter of human ESL culture. Medium to each.Well collect this rinse and transfer to the conical tube pipe at the cells gently in the tube to break up the colony pieces to the desired size. Centrifuge the cells for five minutes at 200 Gs while the human e es cells are being centrifuged. Prepare a new six well meth feeder plate.Label the plate with the appropriate human ES cell information cell line, new passage number and passage date. Aspirate the meth medium from the wells and add one milliliter of PVS to each. Well gently swirl the buffer around the wells and aspirate the PBS wash.Add 1.5 milliliters of human ESL culture. Medium to each well and set the plate aside. After the human ES cells have been centrifuged, bring the tube back to the biological safety cabinet.Aspirate the supernatant. Being careful not to disturb the loosely packed cell pellet. Resuspend the cells in the pellet with enough medium for one milliliter of human ESL culture, medium per new well to be plated.For example, when splitting into six new wells, resus, suspend the pellet in six milliliters of medium. Gently pipette the colonies to break up the pieces to the appropriate size and to evenly disperse the colony pieces. Add one milliliter of the cell suspension to each prepared well of the MEF feeder plate.Place the plate into the 37 degrees Celsius incubator and carefully slide the plate forward to back and slide to side to evenly distribute the cells throughout the well. Allow the cells to attach at 37 degrees Celsius overnight. Differentiated human ES cells can have a range of morphologies, but usually consist of larger and more spread out cells.Differentiation can occur within a colony or between colonies. These are the different morphologies of human ES cultures that can be seen at low magnification under the microscope. A good cell and colony morphology looks like this.Cells are very small, tightly packed and grow in a monolayer. Colonies should have clean and defined edges with little to no differentiation. To maintain healthy human ESL cultures, the cells must be passaged at the optimal time.Colonies that are ready for packaging have almost reached their maximum colony size, but have not begun to fuse together or differentiate. Do not wait until colonies are overgrown to passage them. We've just shown you how to maintain healthy HES cell cultures when following these procedures.Make sure to keep good sterile technique and always keep a backup. So that's it. Thanks for watching and good luck with your experiments.

culture-and-maintenance-of-human-embryonic-stem-cells

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgfβ1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs).  Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

To avoid the limitations associated with the enzymatic or mechanical passaging of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) cultured on feeder cells, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC or hiPSC colonies maintained on a feeder cell layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion.

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

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.

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

Learn to maintain healthy, undifferentiated human embryonic stem cells using daily feeding, manual differentiation removal, and collagenase passaging techniques. This protocol ensures optimal cell viability for research applications.

Culture and Maintenance of Human Embryonic Stem Cells

Explore More Videos