Session - 5-cell separation, cell density and isopycnic sedimentation process in tissue culture

Introduction

  • Title: Animal Cell Culture Technology by Dr. K. Ganesh Prasath

  • Focus: Cell separation, density, and isopycnic sedimentation process in tissue culture.

Principles of Cell Separation

  • Parameters affecting separation:

    • Physical properties: cell density, size.

    • Antibody affinity for ligands on cell surfaces.

    • Unique properties that identify specific cell types.

Categories of Cell Types

Based on Embryonic Development

  • Endoderm Derived:

    • Barrier cells (e.g., epithelial)

    • Hormone-secreting cells

  • Ectoderm Derived:

    • Epithelial cells

    • Nervous system cells

  • Mesoderm Derived:

    • Metabolism/storage cells (e.g., liver, adipocytes)

    • Reproductive system cells

    • Muscle and blood cells

Based on Physiological Function

  • Hormone-Releasing Cells:

    • Thyroid glands

    • Adrenal glands

  • Exocrine Secretory Cells:

    • Salivary glands

    • Pancreas

  • Nervous System Cells:

    • Neurons, glial cells

  • Storage Cells:

    • Lipocytes, adipocytes

  • Extracellular Matrix Cells:

    • Connective tissue fibroblasts, osteoblasts

  • Contractile Cells:

    • Skeletal, cardiac, and smooth muscle cells

Composition of Mammalian Cells

Component

% Total Weight

E. coli

S. cerevisiae

Mammalian Cell

Water

70-80

70

70

70

DNA

3

0.1-0.6

1

1

RNA

20

6-12

4

4

Proteins

50-55

35-60

60

60

Lipids

7-9

4-10

13

13

Density of Mammalian Cells

  • Density Range: 1.055 – 1.110 g/ml

  • Low variation in density for specific cell types:

    • Coefficient of variation for human erythrocytes - 11-15% for size, 0.5% for density.

Isopycnic Centrifugation

  • Technique: Density-based separation, commonly used.

  • Applications: Isolation of stem cell populations from bone marrow, adipose tissue, skeletal muscle.

Density Media

  • Types of Density Medium:

    • Non-toxic, non-viscous at high densities (1.10 g/ml)

    • Examples:

      • Ficoll (GE Healthcare)

      • Histopaque® (Sigma-Aldrich)

      • Lymphoprep and OptiPrep (Sigma-Aldrich)

      • Percoll (GE Healthcare)

Gradient Generation for Cell Separation

  • Techniques:

    • Layer blood cells in plasma over Ficoll-Paque.

    • Utilize a density gradient for separation of viable/non-viable cells, lymphocytes from plasma and erythrocytes, etc.

Tapered Conical Chambers for Gradient Formation

  • Components:

    • High and low-density mediums (1.080 g/ml and 1.020 g/ml)

    • Equipment includes gradient formers, rotating mixers, and delivery tubes.

Gradient Optimization for Cell Separation

  • Factors:

    • Gradient density and separation methods.

    • Selection of appropriate density media (Ficoll, Percoll).

    • Type of gradient (multistep for known cell densities).

    • Centrifugal force and duration to minimize cell damage.

    • Cell position to reduce contamination.

Cell Size and Sedimentation Velocity

  • Sedimentation:

    • Cell size (cross-sectional area) influences sedimentation velocity at unit gravity (1 g).

    • Formula: v = r^2/4 for sedimentation velocity.

    • Useful for separating large differences in cell size or aggregates from single cells.

Centrifugal Elutriation

  • Method Overview:

    • Uses counter-streaming centrifugation.

    • Cells pumped into a chamber during rotation.

    • Achieves equilibrium at different rates based on size and density.

Advantages of Centrifugal Elutriation

  • Efficiency: Fast and gentle process.

  • Cells can be cultured post-separation.

  • Direct applications in laboratory settings.

Magnetic Separation and Sorting Techniques

  • Methods:

    • Using Dynabeads for magnetic cell sorting.

    • MACS technology for positive separation of cell types.

Flow Cytometry

  • Technique: Analyzes and sorts cells based on various parameters (size, immunolabeling).

  • Process Explained:

    • Single cell stream passed through laser detection.

    • Various properties analyzed through light scattering and fluorescence.

Applications of Flow Cytometry

  • Identification of hematopoietic cells.

  • Characterization and separation of various stem cell populations.

Microfluidic Cell Culture

  • Functionality: Enables separation of cells using density gradients without centrifugation.

  • Example: 3D-printed density sorter chip efficiently separates leukocytes from erythrocytes.