Cell Culture and Growing Cells

  • Introduction to Cell Culture

    • Engaged in isolation of DNA from human embryonic kidney cells.

    • Utilized a lysis kit for the process.

  • Cell Lysis & DNA Isolation Process

    • Cells were lysed using a lysis buffer that contains detergent.

    • Noticed that lysis buffer forms bubbles, which is typical behavior due to the detergent.

    • This process is essential for students conducting projects on gene regulation, either up or down.

  • Lysis Buffer Details

    • Detergent in lysis buffer works to break open cells, releasing DNA.

    • Bubble Formation: Normal due to detergent interaction.

    • Centrifugation is used to remove bubbles and separate components effectively.

  • Cell Pellet Preparation

    • Prior to lysis, a cell pellet is formed by trypsinizing adherent cells, then spinning.

    • Important to manage the concentration of lysis buffer to prevent DNA degradation while still lysing cells.

  • Centrifugation Protocol

    • Recommended spin at 8000 RPM for one minute; adjustments made based on cell density if issues arise.

    • Focusing on one variable at a time during troubleshooting is crucial to isolate the problem effectively (either increase RPM or centrifugation time).

Cell Density and Confluency

  • Definition of Cell Density

    • Refers to the number of cells per milliliter in the culture.

    • Excessive cell density can clog the membrane used in the purification process, preventing proper DNA binding.

  • Measuring Viable and Non-Viable Cells

    • Viable cells have intact membranes, while non-viable cells do not and take up stains like Trypan Blue.

    • Methods:

    • Optical Density measurements (limited for viability).

    • Trypan Blue staining for clear identification of viable versus non-viable cells.

    • MTT assay to assess metabolic activity based on color change (purple for live, yellow for dead).

Expression Systems in Cell Culture

  • Types of Expression Systems

    • Prokaryotic Expression System (E. coli):

    • Intracellular protein formation.

    • Mammalian Cells (CHO cells):

    • Extracellular protein formation; lack of cell wall leads to increased risk of shearing during agitation.

  • Confluency

    • A measure of how much surface area of the flask is covered by cells (expressed in percentage).

    • Optimal growth typically occurs at 70-80% confluency; need for splitting occurs as cells approach this limit to prevent nutrient starvation and death from overcrowding.

Culture Conditions and Cell Types

  • Growth Conditions

    • Adherent Cells: Require surface for growth; typically assessed for confluency and require trypsin for detachment.

    • Suspension Cells: Thrive in liquid and can be grown in shaker flasks; preferred in bulk production.

  • Importance of Cell Culture

    • Essential for producing biopharmaceutical proteins and conducting cytotoxicity testing for drug development.

Drug Testing and Cytotoxicity

  • Cytotoxicity

    • Understanding drug concentrations that cells can tolerate is crucial to prevent uncontrolled cell death (necrosis).

    • Emphasis on controlled apoptosis for therapeutic efficacy in cancer treatments.

Bioprocess Scaling and Process Design

  • Scale-up in Cell Culture

    • Need for different vessels based on adherent vs. suspension type; larger reactors or microcarriers for adherent cells.

    • Importance of aeration and agitation for maintaining cell viability and optimal culture conditions.

Challenges in Cell Culture

  • Contamination Control

    • Thorough cleaning and sterilization procedures must be maintained to prevent cross-contamination of cultures.

    • Proper aseptic techniques are critical; the rule of clean side and dirty side in shared hoods.

  • Waste Management

    • Heavy waste generation from single-use disposable cell culture materials; managing costs associated with this waste is a significant aspect of cell culture operations.

Summary of Vital Techniques and Concepts

  • Key Techniques

    • Use of centrifuge to pellet cells; avoid high G-forces to prevent cell damage.

    • Maintaining optimal pH, temperature, and oxygen conditions for cell health and productivity.

  • Sel Culture Laboratory Equipment

    • Biological Safety Cabinets, Incubators, Microcentrifuges, Microscopes, and Storage for reagents must all be sterile and appropriate for the use case.

  • Precautions in Cell Handling

    • Understanding that cells may need to be cryopreserved for future use, with careful considerations to avoid damaging them during this process.

  • Conclusion

    • Comprehensive understanding of culture techniques, cell handling, and bioprocess considerations are essential for successful cell culture practices in a laboratory setting.