Stem cells are revolutionizing biomedical research and healthcare, offering unprecedented potential for regenerative medicine and disease treatment. Whether you are a seasoned researcher or just starting, understanding the nuances of working with stem cells is crucial. This blog aims to simplify various aspects of stem cell culture, from understanding the biology behind stem cells to practical tips for setting up a laboratory and troubleshooting common issues for successful cultivation. 

What Are Stem Cells?

Stem cells are unique cells that can self-renew and differentiate into various specialized cell types. Their unspecialized nature allows them to be versatile, making them invaluable in medical research and therapeutic applications. Stem cells can either divide symmetrically to produce identical daughter cells or asymmetrically to generate specific cell types while retaining their stem cell characteristics. 

Sources of Stem Cells

There are 3 primary sources of stem cells: 

• Embryonic Stem Cells (ESCs): Obtained from the inner cell mass of blastocysts (Embryos). However, ethical considerations of ESCs arise because extracting these cells results in the destruction of the embryo. 

• Induced Pluripotent Stem Cells (iPSCs): Somatic cells reprogrammed to an embryonic-like state. 

• Adult Stem Cells (ASCs): Found in various adult tissues like bone marrow, umbilical cord, fat tissue and even dental pulp. ASCs are commonly known as mesenchymal stem cells (MSCs) with immense therapeutic promise and the use of it is less controversial.

Setting Up a Stem Cell Laboratory

Establishing a stem cell laboratory requires careful planning and adherence to stringent regulations: 

• Laboratory Types
  • One of the crucial decisions you’ll face when establishing a stem cell lab is whether to prioritize a General Laboratory Practice (GLP) or a Good Manufacturing Practice (GMP) facility. This choice will significantly influence your regulatory and safety protocols, ultimately shaping the type of research you can undertake. GLP and GMP are FDA regulations designed to ensure quality and consistency in healthcare products. However, they serve distinct purposes and apply to different stages of product development.
    • Good Laboratory Practice Lab (GLP): Focus on Data Integrity 
      • Typically employed in pre-clinical research, GLP focuses on ensuring data integrity and reliability. It establishes laboratory operations, documentation, and quality control guidelines to ensure that research data is accurate and reproducible.
    • Good Manufacturing Practice (GMP): Focus on Product Safety and Quality
      • Applied to manufacturing processes, GMP mandates strict adherence to quality standards to ensure that products meet safety and efficacy requirements for commercialization. It’s often required for clinical trials and commercial drug production.

• Bio-Safety Measures
  • Bio-safety is a critical aspect of lab setup. Stem cells carry biological risks, especially if they are obtained from donors. Ensure that you have bio-safety level appropriate for the types of cells you’re working with. Always dispose of sharp materials and biological waste following strict biosafety guidelines to minimize contamination risks.
• Record Keeping
  • Meticulous record-keeping is paramount, especially for labs striving to meet GLP or GMP standards. Documenting every step, from batch numbers of culture media to daily experimental results, ensures data integrity and reproducibility. Inconsistent documentation can lead to misunderstandings and unreliable data, jeopardizing the credibility of your research.
  • As Dr. Aon emphasized in our recent webinar “Stem Cell Culture Shock No More”, effective note-taking is a frequently overlooked yet critical aspect of success in translational stem cell research. By meticulously recording details such as the media batch used, the researcher performing the experiment, and even day-to-day results, you lay the foundation for impactful and reproducible research.
  • Looking for a streamlined solution for your lab’s record-keeping needs? Consider our cutting-edge electronic lab notebook, designed to enhance efficiency and collaboration across your research team. 

“Work without Taking Notes Mean No Work” – Dr Ruttachuk Rungsiwiwut (Aon)

• Essential Equipment 
  • Investing in the right laboratory equipment and solutions can go a long way to support you in meeting your laboratory goals. You’ll be surprised that even the most basic lab equipment like your choice can affect your stem cell workflow efficiency and quality. 
    • Centrifuges
      • The type of centrifuge rotor used can significantly influence your results: 
        • Swing-Out Rotors: Suitable for scenarios where gentle handling of cells is required. 
        • Fixed-Angle Rotors: Ideal for sedimenting cells quickly. 
      • Ensure that your choice of centrifuge breaks down the cycle gradually to prevent losing your precious cells in suspension due to the cell pellet resuspending.
    • Laminar Flow Hoods
      • Always use dedicated laminar flow hoods for specific tasks to avoid cross-contamination between different cell cultures.
    •  Choosing the Right Culture Medium
      • The choice of basal medium can affect cell growth and morphology. Options include:
        • DMEM 
        • DMEM Low Glucose 
        • Alpha-MEM
      • Each cell type may require different supplements, such as glucose or glutamine, to support optimal growth. For more specialized applications like clinical trials, serum-free media is often preferred to minimize variables.

Tips for Successful Stem Cell Culture

Culturing stem cells is a delicate art form that requires precision and attention to detail. To achieve optimal results, consider these key factors:

1. Choosing The Right Basal Medium:  
   • Tailor Your Choice: Select a basal medium specifically designed for your stem cell type. Factors like glucose concentration and growth factors play a crucial role in cell viability and function. 
   • Atlantis Bioscience Offerings: Explore our diverse range of basal media, and specialized media for various cell types. 

2. Serum or Serum-Free? 
   • Weigh the Pros and Cons: Using fetal bovine serum (FBS) is common in many labs, but for clinical applications, serum-free conditions are recommended to avoid introducing unknown factors. Serum-containing media provide essential nutrients but can introduce variability. Serum-free media offer more defined conditions, making them ideal for clinical applications. The gradual transition from serum-dependent to serum-free media can help cells adapt without shock. 
   • Leverage Atlantis Bioscience’s STEMGOLD: Our serum-free medium is specifically formulated for mesenchymal stem cells, offering rapid growth and differentiation while maintaining pluripotency. MSCs grown in our STEMGOLD have one of the shortest doubling times, allowing you to grow more cells in a shorter period. 
3. Stem Cells Passaging methods:  
   • Passaging is the process of transferring stem cells from one culture vessel to another to maintain their growth and proliferation. There are two primary methods used for passaging stem cells: 
     • Trypsinization (Enzymatic Passaging): Trypsin, a proteolytic enzyme, is used to detach cells from the culture surface by cleaving the proteins that bind them to the substrate. 
       • Process: 
         • Wash: The culture vessel is washed with a phosphate-buffered saline (PBS) solution to remove any residual media or debris. 
         • Add Trypsin: A trypsin solution is added to the vessel and incubated for a specific time (often a few minutes) to allow the enzyme to detach the cells. 
         • Inactivate: Trypsin activity is neutralized by adding a growth medium containing a serum or growth factor that inhibits trypsin. 
         • Centrifuge: The cell suspension is centrifuged to pellet the cells. 
         • Resuspend: The cell pellet is resuspended in fresh growth medium and transferred to a new culture vessel. 
     • Mechanical Dissociation: Cells are detached from the culture surface by physically disrupting the cell-substrate interactions.
       • Process:  
         • Wash: The culture vessel is washed with PBS. 
         • Detach: Cells are detached using a pipette or a cell scraper. 
         • Centrifuge: The cell suspension is centrifuged to pellet the cells. 
         • Resuspend: The cell pellet is resuspended in a fresh growth medium and transferred to a new culture vessel. 

4. Understanding Morphology 
   • A Visual Cue to Health: Regularly observe your cells under a microscope. Changes in morphology can indicate stress, contamination, or improper culture conditions. Knowing the normal and abnormal morphologies of your cells can help you quickly identify issues. Differentiating cells may change shape, and recognizing these differences can be crucial for timely interventions. 
   • Using Visual Aid to Optimise Your Culture: Use our high-resolution Biotium dyes to visualize and monitor cell morphology effectively. 
5. Cryopreservation for Long-Term Storage: 
   • Preserve Your Cells: Develop robust cryopreservation protocols to store stem cells for extended periods. Read here to find out how to achieve successful cryopreservation for MSCs.

By carefully considering these factors and leveraging Atlantis Bioscience’s comprehensive range of lab solutions, you can optimize your stem cell culture and achieve exceptional results.

Common Challenges in Stem Cell Culturing and Solutions

Cell culture can be a delicate balancing act, with various factors influencing the success of your experiments. Let’s explore some common pitfalls and strategies to overcome them:

• Heterogeneous Populations 

Sometimes, isolated cells from tissues can result in a heterogeneous population. Techniques like magnetic-activated cell sorting (MACS) can help isolate more homogeneous cell populations from heterogeneous mixtures. When working with stem cells that have a smaller population, explore other techniques to purify your cells. 

• Cross-Contamination 

Cross-contamination is a frequent issue, especially in labs handling multiple cell types. Strictly adhere to sterilization protocols and avoid using the same pipettes for different cultures. 

• Mycoplasma Contamination 

Mycoplasma is a common contaminant that can severely affect cell cultures. Regularly screen for mycoplasma by using colourimetric or PCR-based assays to ensure your cultures are contamination-free. 

• Controlling Cell Density 

Consistency in cell seeding density is crucial. Over-confluent or overly sparse cultures can both lead to suboptimal cell growth. Regularly monitor and record the growth rate to optimize cell density. 

Advanced Techniques and Applications of Stem Cells 

• Organoid Cultures 

By using embryonic or iPS cells, you can grow organoids that mimic the architecture and functionality of real organs. This is particularly useful for studying disease models and drug response. 

• Preclinical Trials 

Leveraging stem cells in preclinical trials can accelerate the development of therapies. Using stem cells derived from the same species as the host organism minimizes immunogenic reactions. 

• Genetic Screening 

For clinical-grade cultures, genetic screening of stem cells for abnormalities using various molecular techniques ensures the safety and efficacy of cell-based therapies. 

• Exosome Production 

Stem cells can be engineered to produce exosomes loaded with therapeutic cargo. These exosomes can then be used in various treatments, ranging from regenerative medicine to cancer therapy.

Stem cell research holds immense promise, but it also comes with its own set of challenges. From understanding the basic biology of stem cells to setting up a laboratory and optimizing culture conditions, each step is crucial for successful outcomes. Regularly updating lab practices, maintaining stringent bio-safety measures, and meticulously documenting all processes will help you navigate this complex field more effectively.

By staying informed and adopting the best practices highlighted in this blog, you can ensure that your stem cell research is not only effective but also safe and reproducible. Whether you’re conducting basic research or aiming for clinical applications, these guidelines will provide a strong foundation for your work in stem cell science.