Presentation is loading. Please wait.

Presentation is loading. Please wait.

Cell isolation: Procedure and Troubleshooting

Published byBrian Peters Modified over 9 years ago

Similar presentations

Presentation on theme: "Cell isolation: Procedure and Troubleshooting"— Presentation transcript:

1 Cell isolation: Procedure and Troubleshooting
Sepideh Khoshnevis

2 Why do we need to isolate these cells?
We need to have cells in culture in order to gather data on HSP expression and cell death Cells from normal canine tissue are not commercially available This process is called primary culture There are many challenges as they would be explained

3 Procedure

4 Culture of epithelial cells
Acquisition of tissue Isolation of cells Primary culture Subculture Freezing and thawing cells

5 Acquisition of tissue Quality of tissue
Viability Tissue transportation to the laboratories Media HBSS Saline Cell appropriate media Temperature Oxygenation

6 Isolation of cells Enzymatic method Washing steps Mincing step
Centrifugation RCF duration Mincing step Temperature Media size Collagenase step Objective is to dissolve/digest intercellular matrix without damaging the cells Composition of collagenase solution This is potentially the most stressful step on the cells Rocking step Oscillations per minute

7 Primary culture issues
Coated vs non-coated surface Collagen I Collagen IV Media that is friendly to the cells No guidelines are available to define the best/appropriate media composition Small changes in composition can have large consequences for cell survival

8 Example media compositions

9 Subculture to get enough cells to conduct experiments
Trypsinization step to remove cells from culture surface Trypsin concentration Duration Trypsin inactivation Incubation step Temperature CO2 concentration

10 Troubleshooting

11 Non-adherent cells Surface coating of the slide
Choice of ECM elements Coating protocol Different concentration Drying procedure sterilization Use of elements that promote surface adhesion Testosterone

12 Non-adherent cells The cells are apoptotic Trypsinization step
0.05% trypsin VS 0.25% trypsin Incubation condition CO2 concentration Acquisition of tissue Transport of whole tissue VS tissue pieces Composition of transport media

13 Non-adherent cells Choice of media Toxicity Composition
Isolated cell types Stromal VS epithelial Characterization of epithelial cells

14 Characterization of epithelial cells
Morphology Immunostaining Anti-cytokeratin antibody Anti-vimentin antibody Anti-SMA antibody

15 Cos7 in epithelial environment
Day 3 Day 1 Day 2 Day 4 Day 5 Day 6

16 Prostate stromal cells
Day 1 Day 2 Day 3 Day 4

17 Conclusion Still can not isolate prostatic epithelial cells successfully and satisfactorily Possible causes Problem with collagenase step Lack of essential nutrients in collagenase solution Problem with incubation Too high CO2 concentration Amongst a very large number of coupled variables there are no other apparent variable contributing to the problem at hand

18 Future steps Do another cell isolation with the following changes
Change of the protocol Change of the collagenase solution adding serum and media to the solution Using lower concentration of collagenase Longer incubation in collagenase solution Use lower concentration of trypsin Improve incubation condition By correcting the CO2 concentration Cell characterization at the end of isolation By performing immunofluorescence studies on the cell smear using anti-cytokeratin, anti-SMA and anti-vimentin antibodies No final success yet but significant progress

Download ppt "Cell isolation: Procedure and Troubleshooting"

Similar presentations

Introduction to Physiology

Introduction to Physiology
Primary Culture of Chick Myocyte. Establishment of primary cultures from various sources Consider:  Whether to use normal or tissue derivative tissue.

Primary Culture of Chick Myocyte. Establishment of primary cultures from various sources Consider:  Whether to use normal or tissue derivative tissue.
ViahanceTM: Dead Cell, Stripped Nuclei and Free Oligonucleotide Removal Kit Instructions ViahanceTM dead cell removal kit enhances the viability ratio.

ViahanceTM: Dead Cell, Stripped Nuclei and Free Oligonucleotide Removal Kit Instructions ViahanceTM dead cell removal kit enhances the viability ratio.
When can you use an antibody to find another antibody?

When can you use an antibody to find another antibody?
IMMUNOLOGY LABORATORY PBMC ISOLATION SOP by Kizza D Martin Ssemambo

IMMUNOLOGY LABORATORY PBMC ISOLATION SOP by Kizza D Martin Ssemambo
Mammalian Cell Culture. What is cell culture, exactly?  Cells, previously growing in a human or animal modified to grow in plastic or glass In the body.

Mammalian Cell Culture. What is cell culture, exactly?  Cells, previously growing in a human or animal modified to grow in plastic or glass In the body.
Islamic University _Gaza Department of Biotechnology

Islamic University _Gaza Department of Biotechnology
Islamic University _Gaza Faculty of science Department of Biotechnology By: Mahmoud W. El-Hindi 2013-2014 1 By: Mahmoud El-Hindi.

Islamic University _Gaza Faculty of science Department of Biotechnology By: Mahmoud W. El-Hindi By: Mahmoud El-Hindi.
Tissue Culture Methodology. Historical Perspective Early 20 th Century Technique Antibiotics and Laminar Flow Hoods Allow for Tissue Culturing to Grow.

Tissue Culture Methodology. Historical Perspective Early 20 th Century Technique Antibiotics and Laminar Flow Hoods Allow for Tissue Culturing to Grow.
400x100x200x ABC Epithelial cell Attached bacteria Figure 1 E. coli attachment to human bladder epithelial cells. (A) After two hours, 29214 has begun.

400x100x200x ABC Epithelial cell Attached bacteria Figure 1 E. coli attachment to human bladder epithelial cells. (A) After two hours, has begun.
Microbiology and Serology

Microbiology and Serology
The Effects of Ibuprofen On Stem Cell Development Kevin Pfau Central Catholic High School Grade 11.

The Effects of Ibuprofen On Stem Cell Development Kevin Pfau Central Catholic High School Grade 11.
Kenneth M. Yamada & Edna Cukierman. Introduction 2D cell cultures Studies in standard cell culture have produced many important conceptual advances Nevertheless,

Kenneth M. Yamada & Edna Cukierman. Introduction 2D cell cultures Studies in standard cell culture have produced many important conceptual advances Nevertheless,
Primary Culture Chapter 12.

Primary Culture Chapter 12.
Structure, function and growth of prokaryote and eukaryote cells (iii) Differentiation of cells into tissues and organs. (iv) Cell and tissue culture –

Structure, function and growth of prokaryote and eukaryote cells (iii) Differentiation of cells into tissues and organs. (iv) Cell and tissue culture –
1 Autologous Liver Cell Transplantation A new approach to liver disease therapy Presentation at ESAO Conference, September 6 th 2007, Krems Prof. Toni.

1 Autologous Liver Cell Transplantation A new approach to liver disease therapy Presentation at ESAO Conference, September 6 th 2007, Krems Prof. Toni.
Chapter 20: Introduction to Animal Physiology

Chapter 20: Introduction to Animal Physiology
Practical Blood Bank Lab 4 Weak D testing (Du).

Practical Blood Bank Lab 4 Weak D testing (Du).
Basic technique Training and and Practice  Pipetting and transfer of fluid  Observation of cultured cells  Aseptic technique: preparation of mediums.

Basic technique Training and and Practice  Pipetting and transfer of fluid  Observation of cultured cells  Aseptic technique: preparation of mediums.
Proteomics Module Day 1 Tech talk. Experiment: Yeast protein expression changes caused by H 2 O 2 exposure. ► 2 Control groups (A and B): nothing added.

Proteomics Module Day 1 Tech talk. Experiment: Yeast protein expression changes caused by H 2 O 2 exposure. ► 2 Control groups (A and B): nothing added.

Similar presentations

Ads by Google