Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Hongzi Chen; Pan Huang; Yan Zhang

doi:10.3791/63585

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

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Biology

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JoVE Journal Biology

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

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07:49 min

June 2, 2022

DOI: 10.3791/63585-v

Hongzi Chen¹, Pan Huang¹, Yan Zhang¹

¹MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences,Sun Yat-sen University

Overview

This study details a serum-free culture method for enriching adult lacrimal gland stem cells, facilitating their differentiation into acinar and ductal-like cells. The protocol emphasizes the capability of these cells for long-term expansion and regenerative potential, providing a valuable model for lacrimal gland repair.

Key Study Components

Research Area

Stem cell biology
Lacrimal gland regeneration
Tissue engineering

Background

Lacrimal gland stem cells are essential for tear production and ocular surface health.
Current methods for culturing these cells often involve serum, which presents limitations.
Establishing a serum-free system can enhance cell viability and differentiation.

Methods Used

Isolation and culture of lacrimal gland stem cells from euthanized BALB/c mice.
Use of enzymatic dissociation with dispase and collagenase to obtain single-cell suspensions.
In vitro culture in a newly developed lacrimal gland stem cell medium.

Main Results

Long-term expansion and differentiation of lacrimal gland stem cells were successfully achieved.
Monitoring of cell spheres indicated proliferation and differentiation capabilities.
Successful induction of lacrimal gland structures upon orthotopic transplantation in mice.

Conclusions

The study demonstrates a successful protocol for culturing and differentiating lacrimal gland stem cells.
This work is fundamental for advancing research into lacrimal gland repair and potential treatments for associated diseases.

Frequently Asked Questions

What is the relevance of lacrimal gland stem cells?

Lacrimal gland stem cells are crucial for maintaining tear production and overall ocular health.

How does the serum-free method benefit stem cell culture?

A serum-free method reduces variability and enhances the ability of stem cells to grow and differentiate optimally.

What are the main findings regarding differentiation?

The protocol led to efficient differentiation into acinar and ductal-like cells, with successful restoration of lacrimal structures.

What techniques were used to analyze the stem cells?

Techniques included microscopy for monitoring cell spheres and histological staining to evaluate differentiation.

Can this method be applied to other types of stem cells?

While the protocol is specific to lacrimal gland stem cells, the fundamental principles can potentially be adapted for other types of tissue stem cells.

What is the significance of the in vivo results?

The in vivo results indicated successful integration and function of the implanted cells, suggesting potential therapeutic applications.

Are there any limitations to this study?

Further research is needed to understand the long-term effects and optimization of the protocol for clinical applications.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

This protocol provides an advantageous strategy for enriching lacrimal gland stem cells for the regeneration and reconstruction of lacrimal gland. Lacrimal gland stem cells show long-term expansion capacity and the potential of differentiation. The main advantage of this technique is that the culture medium for lacrimal gland stem cells is serum-free, which shows the enormous value for lacrimal gland repair Begin by obtaining a euthanized six-to eight-week-old BALB/c male mouse and cutting the skin behind the ear to expose the lacrimal gland and the connective tissue around it.

Peel off the connective tissue by blunt dissection with the help of tweezers and remove the lacrimal gland. Immerse the lacrimal glands in a six-centimeter dish with four milliliters of 75%ethanol for 10 seconds and immediately rinse with 10-millimolar PBS solution twice. Cut the lacrimal glands into small fragments of about one millimeter cubed.

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