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  2. 2. UNDER THE GUIDANCE OF <ul><li>INTERNAL GUIDE : L.G. MOHANA DIVYA </li></ul><ul><li>Lecturer </li></ul><ul><li> Department Of Biotechnology </li></ul><ul><li> KSR College Of Technology </li></ul><ul><li>EXTERNAL GUIDE : DR. ALEEM KHAN </li></ul><ul><li>Centre For Liver Research And Diagnostics Hyderabad. </li></ul>
  3. 3. INTRODUCTION <ul><li>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. </li></ul><ul><li>There can be two sources of stem cells- Autologous and Allogenic. </li></ul><ul><li>Autologous embryonic stem cells generated through therapeutic cloning and highly plastic stem cells from the umbilical cord blood or bone marrow from candidates. </li></ul><ul><li>Allogenic stem cells can be derived from marrow, peripheral blood, cord blood, family donors or HLA typed donors . </li></ul>
  4. 4. PROPERTIES OF STEM CELLS <ul><li>1.determining precisely how stem cells remain unspecialized and self renewing for many years; and </li></ul><ul><li>2.identifying the signals that cause stem cells to become specialized cells. </li></ul>
  5. 5. TYPES OF STEM CELLS <ul><li>EMBRYONIC STEM CELL </li></ul><ul><li>ADULT STEM CELL </li></ul><ul><li>FETAL LIVER STEM CELL </li></ul><ul><li>UMBILICAL CORD STEM CELL </li></ul><ul><li>PANCREATIC STEM CELL </li></ul>
  6. 6. OBJECTIVE <ul><li>To Isolate stem cells from human foetal liver with the help of markers Alfa feto protein, cytokeratin 18 & Albumin. </li></ul><ul><li>To characterize the isolated stem cells using different methods. </li></ul>
  7. 7. STEPS INVOLVED <ul><li>FOETAL DISSECTION </li></ul><ul><li>LIVER EXTRACTION </li></ul><ul><li>DIGESTION OF LIVER </li></ul><ul><li>STEM CELL ISOLATION USING MACS </li></ul><ul><li>CELL COUNTING - HAEMOCYTOMETER </li></ul><ul><li>CELL VIABILITY ASSAY </li></ul><ul><li>IMMUNOCYTOCHEMISTRY </li></ul>
  8. 8. CONTINUE… <ul><li>MICROTOXICITY ASSAY </li></ul><ul><li>RNA ISOLATION FROM STEM CELLS </li></ul><ul><li>QUANTIFICATION OF RNA </li></ul><ul><li>REVERSE TRANSCRIPTION </li></ul><ul><li>RT PCR </li></ul><ul><li>AGAROSE GEL ELECTROPHORESIS </li></ul>
  9. 9. LIVER CELL EXTRACTION <ul><li>Homogenization is done with collagenase 4 for complete extraction of liver cells. </li></ul><ul><li>Cell washing is done with Hanks buffer 3-4 times. </li></ul>
  10. 10. ISOLATION OF STEM CELLS <ul><li>Isolation of stem cells can be done with MACS and FACS. </li></ul><ul><li>MACS - Magnetic associated cell sorter and FACS- Fluorescent activated cell sorter </li></ul><ul><li>MACS is commonly used as fluorescent can cause damage to the cells during isolation. </li></ul>
  11. 11. MAGNETIC ASSOCIATED CELL SORTER <ul><li>1. Take the standardized cell sample containing 5 million cells in sterile eppendroff. </li></ul><ul><li>2. Add 2 µl of FCR blocking reagent to the sample and same volume of AFP antibody into the same eppendroff. </li></ul><ul><li>3. Incubate for 30-45 mins at temperature of 4 °c. </li></ul>
  12. 12. CONTINUE… <ul><li>4. MACS with LS max column is used for separation of stem cells containing AFP+ and AFP- cells . </li></ul><ul><li>5. The separation of +ve and –ve cells is due to the magnetic field applied and the binding of specific antibodies with the cells. </li></ul><ul><li>6. The cells which do not bind with the specific antibody elute first and are referred as AFP- cells. </li></ul>
  13. 13. CONTINUE… <ul><li>7 . The remaining cells in the column are extracted by adding Hanks buffer and with the help of pressure syringe. </li></ul><ul><li>8. The extracted cells are referred as AFP+ cells. </li></ul><ul><li>9. The cells are washed with Hanks Buffer 2-3 times. </li></ul><ul><li>10. The cells are preserved in 4 °c with the addition of Hanks buffer . </li></ul>
  14. 14. CELL COUNTING <ul><li>Cell counting is the process of measuring total number of cells per ml. </li></ul><ul><li>It is done with the help of Haemocytometer </li></ul><ul><li>The cells are mixed with trypan blue dye in the ratio 1:10 such as dye and cells respectively. </li></ul><ul><li>The cells are made into exact concentration and placed on the square slide covered with cover slip. </li></ul>
  15. 15. CONTINUE… <ul><li>The slide is placed on the microscope and observed for total number of cells. </li></ul><ul><li>The calculation is done with the formula </li></ul><ul><li>number of cells = number of * dilution per cubic mm cells counted factor </li></ul>
  16. 16. RESULTS <ul><li>CELL COUNTING : </li></ul><ul><li>Total number of cells in small square = 43 </li></ul><ul><li>Total number of cells in large square = 43*16= 688 </li></ul><ul><li>Total number of cells </li></ul><ul><li>counted = 688*4= 2752 </li></ul>
  17. 17. APPLICATIONS <ul><li>Stem cells are used in blood transfusions. </li></ul><ul><li>Used in organ transplantation. </li></ul><ul><li>Used in cancer studies. </li></ul><ul><li>Used in diagnostic of diseases such as Parkinson's, diabetes etc. </li></ul>
  18. 18. THANK YOU