From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

Ivan Khvorostov; Jin Zhang; Michael Teitell

doi:10.3791/831

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

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From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

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11,879 Views

06:40 min

June 9, 2008

DOI: 10.3791/831-v

Ivan Khvorostov¹, Jin Zhang¹, Michael Teitell¹

¹David Geffen School of Medicine,University of California, Los Angeles

Overview

This video demonstrates the maintenance of human embryonic stem cells (hESCs) in feeder cell-free conditions, including continuous passaging and confirmation of pluripotency through immunofluorescence microscopy.

Key Study Components

Area of Science

Stem Cell Biology
Cell Culture Techniques
Pluripotency Assessment

Background

Human embryonic stem cells (hESCs) are crucial for developmental biology and regenerative medicine.
Feeder cell-free culture conditions can simplify the maintenance of hESCs.
Pluripotency is a key characteristic of hESCs, essential for their use in research.
Immunofluorescence microscopy is a common method to assess pluripotency.

Purpose of Study

To demonstrate the techniques for maintaining hESCs in feeder cell-free conditions.
To show the process of continuous passaging of hESCs.
To confirm the pluripotency of hESCs using immunofluorescence microscopy.

Methods Used

Changing culture media every 24 hours.
Warming media to 37 degrees Celsius before use.
Adding FGF to the culture media at a specified concentration.
Using immunofluorescence microscopy for pluripotency confirmation.

Main Results

Successful maintenance of hESCs in feeder cell-free conditions.
Effective continuous passaging techniques demonstrated.
Confirmation of hESC pluripotency through immunofluorescence microscopy.
Standardized protocols for media preparation and handling.

Conclusions

Feeder cell-free conditions are viable for hESC culture.
Continuous passaging can be effectively performed under these conditions.
Immunofluorescence microscopy is a reliable method for assessing pluripotency.

Frequently Asked Questions

What are human embryonic stem cells?

Human embryonic stem cells (hESCs) are pluripotent cells derived from early-stage embryos, capable of differentiating into various cell types.

Why use feeder cell-free conditions?

Feeder cell-free conditions simplify the culture process and reduce variability in hESC maintenance.

How often should the culture media be changed?

The culture media should be changed every 24 hours to maintain optimal growth conditions for hESCs.

What is the role of FGF in hESC culture?

FGF (Fibroblast Growth Factor) is added to the culture media to support the growth and maintenance of hESCs.

How is pluripotency confirmed in hESCs?

Pluripotency is confirmed using immunofluorescence microscopy, which detects specific markers associated with pluripotent cells.

What temperature should the media be warmed to before use?

The media should be warmed to 37 degrees Celsius before it is added to the hESC culture.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

To maintain the human embryonic stem cell culture, the culture media must be changed every 24 hours. To do this, start with an embryonic stem cell media stock bottle stored at four degrees Celsius. Take out the amount of solution needed, which is usually about 20 milliliters per six.

Well plate and place that amount in a 50 milliliter Falcon tube and warm the tube with contents to 37 degrees Celsius in a water bath. Once the media is warmed, add the FGF, which has been stored at four degrees Celsius to a final concentration of 10 nanograms per mil. Be sure to remember to put the BFGF stock back at four degrees Celsius immediately after use.

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