EGFP Labeled Immortalized Podocyte Culture (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Racheal Jappert; Jeffrey Pippin; Ying Zheng; Stuart Shankland

Keywords

[‘mouse’, ‘human’, ‘Adult podocytes’]

Subjects

[‘Cell culture’, ‘Tissue culture’]

Release Date

2017-05-25

Abstract

Describes the procedure for maintaining mouse podocytes (isolated from a defined source) in culture.

Introduction

EGFP immortalized podocytes have been isolated from podocyte reporter mice (Nephrin FLP EGFP FRT) crossed with ImmortoMice (containing the interferon driven thermo-sensitive large T antigen from SV40). The cells are from a heterogeneous defined source of adult glomerular podocytes, as they were fluorescent assisted cell sorted for EGFP expressing cells.

Procedure

Collagen Coating Plates

1. Use Bovine Collagen I Santa Cruz Biotechnology, 10410 Finnell Street, Dallas, TX 75220 - 800-457-3801 a. Catalog # sc-29009 b. 30 milligrams c. Store at 4 degrees Celsius
2. Mix 0.2ml of Collagen I with 15ml Sterile dH20 - both stored in door of Tissue Culture Refrigerator E-158.
3. Use Primaria culture ware, and apply solution, then remove so that the plastic has a thin coating of the solution. (save the plastic bag the culture ware came in and keep it in the hood until finished)
4. Allow the plates to dry in the biosafety cabinet with lids open- takes about 1 hour.
5. Re-apply the solution for a second coat.
6. Allow to dry as above.
7. Label plates so that it is clear that they have been coated and the date.
8. Place plates back in the sterile bag they came in and seal tightly. Store @ 4 degrees Celsius

Growth Media

(Note Growth Media is different from B6 Immortalized Podocytes)

• 500ml RPMI 1640 (Fisher Thermo SH3060501) Hyclone Classical Liquid Media without L glutamine, without phenol red
• 50ml Nu-Serum IV 9% (Fisher CB-55004, Corning 355504)
• 5ml Pen/Strep (Biosource 15140-163)
• 5ml Sodium Pyruvate (Fisher SH3023901)
• Combine and Sterile Filter through 0.22um filter flask.
• Store @ 4 degrees Celsius

Growth Permissive Conditions:

1. Collage Coated Plates
2. Add 50units/ml mouse interferon-γ (Roche #11276905001)
3. Grow at 33 degrees Celsius and passage upon confluence. (Do NOT allow to over grow)

Growth Restrictive Conditions:

1. Primaria Plastic Plate, no collagen coating (this is to encourage podocytes in differentiated conditions to produce their own native matrix as podocytes)
2. No interferon-γ
3. Grow at 37 degrees Celsius (monitor your incubator with a thermometer separate from the incubators electronic controls to make certain these temperatures are correct)

What you should expect to see:

Podocytes will increase in size and growth will slow over the first 6 days. Cells can be re-plated for experiments on day 3 or 4 of growth restriction at a density of 6000-10,000 cells/cm2, it is not always ideal to re-plate cells later, although we have had some success with this. Cells will continue to increase in size and assemble a complex actin cytoskeleton. They will initially develop many processes. As they differentiate the cells will become more circular and will assemble cortical actin. Podocytes can be considered fully differentiated by day 14.

Things you should watch for:

Immortalized podocytes in growth permissive conditions are particularly prone to spontaneous transformation. Spontaneous transformation occurs when cells are allowed to grow at high density. For this reason, do not allow cells to overgrow in growth permissive conditions and do not over-plate cells!

Make certain cells are dispersed well and no clumps are present during passaging, as this introduces highly confluent foci that can transform. Use of a cell strainer can help with this. An indication transformation has occurred is if you see populations of cells that do not growth arrest and differentiate in restrictive conditions.

Use 50 units/ml of INF. Lower doses can lead to the selection of subpopulations that will not require INF for proliferation. These populations will eventually dominate cultures and will not growth arrest or differentiate in growth restrictive conditions.

Expand and freeze down many stock vials of lower passage cells upon arrival. If cultures become dominated by spontaneously transformed populations, discard the cultures and revert back to your supply stocks.

Lastly, monitor the temperature of your incubators periodically with a thermometer to make certain that temperatures are correct for growth permissive and growth restrictive conditions.

Consortium

(Re)Building a Kidney (RBK) Consortium