ISOLATION OF HUMAN FETAL LIVER STEM CELLS WITH ALFA

FETO PROTEIN, CK18 & ALBUMIN

Presented by S. EZHIL MANGAI

UNDER THE GUIDANCE OF

INTERNAL GUIDEINTERNAL GUIDE : : L.G. MOHANA DIVYAL.G. MOHANA DIVYA Lecturer Lecturer

Department Of BiotechnologyDepartment Of Biotechnology KSR College Of Technology KSR College Of Technology

EXTERNAL GUIDEEXTERNAL GUIDE : : DR. ALEEM KHANDR. ALEEM KHAN Centre For Liver Centre For Liver

Research And Diagnostics Research And Diagnostics Hyderabad. Hyderabad.

INTRODUCTION

• Stem cells are primal cells common to all multicellular organisms that retain the ability to renew themselves through cell division and can be differentiated into wide range of specialized cell types.

• There can be two sources of stem cells- Autologous and Allogenic.

• Autologous embryonic stem cells generated through therapeutic cloning and highly plastic stem cells from the umbilical cord blood or bone marrow from candidates.

• Allogenic stem cells can be derived from marrow, peripheral blood, cord blood, family donors or HLA typed donors.

OBJECTIVE

To isolate the fetal liver Cells from the fetus by two step collagenase method.

Separation of CD34+ cells by magnetic activated cell sorting method.

Characterization of isolated cells by polymerase chain reaction by using cell markers namely ALB, AFP, CK18.

VIABILITY ASSAY

The cell viability test was performed in total cells and in MACS sorted cells by dye exclusion method.

Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye

The dead cells intake dye which results in blue colour. Whereas the viable cells remain colorless.

IMMUNOCYTOCHEMISTRY To characterize the isolated stem cells. It visualizes the sub cellular localization of antigen in

isolated stem cell populations. Immunocytochemistry refers to a process of localizing

proteins in the cells exploiting the binding of antibodies to antigen.

The visual marker may be a fluorophore (like FITC, rhodamine), colloidal metal, hapten, radioactive marker or more commonly for light microscopy an enzyme. Ideally, maximal signal strength along with minimal background or non-specific staining is required to give optimal antigen demonstration.

PROCEDURE

Take 5 million cells and add 10 µl of diluted primary antibody

↓ Incubate for 2-3 hrs

↓ Wash the cells by applying centrifugation at

1000 rpm for 2 mins at 4°c

↓ Remove the supernatant and add 10 µl of

diluted secondary antibody

↓ Incubate for 1-2 hrs

↓ prepare cell smear on slide and fix using

formaldehyde

↓ Observe the cells under fluorescent

microscopy

Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells.

A solubilization solution (usually either

dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in dilute hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution.

The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The absorption maximum is dependent on the solvent employed.

PROCEDURE

1.5 ml sample is added to 1 ml of MTT reagent

↓ Incubation for 2-3 hrs at room temperature

↓ Add 1 ml of isopropanol

↓ Vortex the sample

↓ centrifuge at 3000 rpm for 4 mins

↓collect the supernatant and calculate OD

at 570 nm

↓ With the readings plot a graph for absorbance vs. activity of cells.

RESULTS

VIABILITY ASSAY

Number of cells in small square =30

Number of viable cells =14

Number of dead cells =16

Number of cells in =30*16 large square =480

Total number of cells =480*4

=1820

Number of viable cells in =14*16

large square =224

Total number of viable cells =224*4

=896

Percentage of viable cells = total no. of viable

cells * 100 total no. of cells = 896 * 100 1820 = 49.23%

IMMUNOCYTOCHEMISTRY

MTT ASSAY

Absorbance of MTT Formazan, the Standard Procedure to Access Cell Activity:

Concentration of cell activity (µg/ml)

OD at 580 nm

Blank 0.0000

50 0.2621

100 0.4421

150 0.6240

200 0.8398

250 0.9945

300 1.1436

Estimation of cell activity by MTT assay: 

Percentage of activity (µg/ml)

OD at 540 nm

35 0.1349

39 0.1790

WORK TO BE DONE

RNA expression studies RNA isolation Reverse transcription PCR Polymerase chain reaction Agarose gel electrophoresis

THANK YOU