Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Yun-Ui Bae; Hoon-Ki Sung; Jae-Ryong Kim

doi:10.3791/55035

Коллекция Serum- и фидерных свободной эмбриональных стволовых клеток мыши-кондиционированная сред...

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Developmental Biology

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JoVE Journal Developmental Biology

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Коллекция Serum- и фидерных свободной эмбриональных стволовых клеток мыши-кондиционированная среда для подхода бесклеточный

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09:32 min

January 8, 2017

DOI: 10.3791/55035-v

Yun-Ui Bae¹, Hoon-Ki Sung^2,3, Jae-Ryong Kim¹

¹Department of Biochemistry and Molecular Biology and Smart-aging Convergence Research Center, College of Medicine,Yeungnam University, ²Physiology and Experimental Medicine Program,The Hospital for Sick Children Research Institute, ³Department of Laboratory Medicine and Pathobiology,University of Toronto

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Overview

This protocol outlines a method for collecting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) under serum and feeder-free conditions. It aims to explore the anti-senescence properties of soluble factors secreted by mESCs for potential therapeutic applications in aging-related diseases.

Key Study Components

Area of Science

Cell Biology
Stem Cell Research
Aging and Regenerative Medicine

Background

Mouse embryonic stem cells (mESCs) can secrete factors that may influence aging.
Conditioned medium from mESCs can be used in a cell-free therapeutic approach.
Understanding the biological functions of mESC-CM is crucial for developing treatments for age-related diseases.
This protocol is designed to be performed in a biological safety hood to ensure safety during cell handling.

Purpose of Study

To collect and characterize mESC-CM under serum and feeder-free conditions.
To investigate the anti-senescence activity of factors secreted by mESCs.
To develop a safe, cell-free therapeutic approach for aging-associated diseases.

Methods Used

Culture mouse embryonic fibroblasts (MEFs) in a controlled environment.
Treat MEF cultures with mitomycin C to inhibit proliferation.
Collect conditioned medium after washing and detaching the cells.
Characterize the biological functions of the collected medium.

Main Results

The protocol successfully collects mESC-CM under defined conditions.
Insights into the anti-senescence properties of mESC-CM are obtained.
Potential applications in treating aging-related diseases are highlighted.
Demonstration of the procedure by Dr. Yun-Ui Bae provides practical guidance.

Conclusions

The collection of mESC-CM is feasible and informative.
Further research may lead to novel therapies for aging-related conditions.
Cell-free approaches using mESC-CM could be a safe alternative in regenerative medicine.

Frequently Asked Questions

What is mESC-CM?

mESC-CM refers to the conditioned medium derived from mouse embryonic stem cells, which contains soluble factors that may influence cellular processes.

Why is a cell-free approach important?

A cell-free approach reduces the risks associated with cell transplantation and can provide a safer therapeutic option for patients.

How does mitomycin C affect MEFs?

Mitomycin C is used to inhibit the proliferation of MEFs, allowing for the collection of conditioned medium without interference from dividing cells.

What are the potential applications of mESC-CM?

mESC-CM may be used in therapies for aging and age-associated diseases by leveraging its anti-senescence properties.

Who demonstrated this protocol?

Dr. Yun-Ui Bae, a research professor, demonstrated the protocol in a laboratory setting.

What safety measures are taken during the procedure?

The procedure is conducted in a biological safety hood to ensure safety while handling cell cultures.

Этот протокол обеспечивает способ сбора эмбриональных стволовых клеток мыши (МЕСК) -conditioned среда (МЕСК-СМ), полученные из сыворотки (эмбриональной бычьей сыворотки, ФБС) - и фидерные (мышиные эмбриональные фибробласты, MEFs) -свободных условия для ячейки -бесплатно подход. Это может быть применимо для лечения старения и возрастного заболеваний.

Общие цели данного протокола заключаются в сборе кондиционированной среды из эмбриональных стволовых клеток мыши в условиях беспитательных культур сыворотки и фидера и в характеристике биологических функций этой среды. Эта процедура дает представление об антистаресцентной активности растворимых факторов, секретируемых из эмбриональных стволовых клеток мыши, для разработки безопасного и бесклеточного терапевтического подхода к возрастным заболеваниям. Демонстрировать эту процедуру будет доктор Юн-Уи Бэ, научный сотрудник моей лаборатории.

В биологической защитной капюшоне обрабатывайте эмбриональный фибробласт мыши или культуру клеток MEF 20 миллилитрами среды MEF с добавлением митомицина C в 15-сантиметровой чашке для клеточной культуры в течение двух часов при температуре 37 градусов Цельсия и 5% углекислого газа. В конце инкубации трижды промойте клетки PBS и отделите их тремя миллилитрами трипсина ЭДТА на три минуты при температуре 37 градусов Цельсия. Нейтрализация трипсина шестью миллилитрами свежей среды MEF, когда клетки поднялись со дна пластины.

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