Best practices for naming, receiving, and managing cells in culture

Yvonne Reid, Consultant, Retiree of ATCC

Cell lines are widely used in biomedical research laboratories as tools or models for various diseases.  It is now estimated that more than one third of the most commonly used cells lines are misidentified.  This increased use of misidentified, including microbial contaminated and poor quality cells, is largely due to poor cell culture practices, and has led to spurious and irreproducible results.

In 2015, a working group of scientists with expertise in various disciplines was assembled to write a series of articles on Best Practices in Cell Culture.  The objective of this project was to produce a series of in-depth publications in best practices, from receiving a cell line to processing, application, storage and distribution. The members of the working group consisted of Dr. John M. Baust (CPSI Biotech), Dr. Gertrude Case Buehring (UC Berkeley), Dr. Lia H. Campbell (Tissue Testing Technologies, LLC), Dr. Eugene Elmore, Dr. John W. Harbell (JHarbell Consulting, LLC), Dr. Ray W. Nims (RMC Pharmaceutical Solutions, Inc.), Dr. Paul Price (Cell Culture Media Consultant), Dr. Yvonne A. Reid (ATCC) and Frank Simione (ATCC).

The Best Practices in Cell Culture: an Overview (Baust et al. Best Practices in Cell Culture: an Overview. In Vitro Cell Dev Biol-Animal 53:669-672, 2017) describes a series of articles that provide an unmet need for information on best practices in animal cell culture. The style and content of the articles are suitable for graduate students, laboratory directors, post-doctoral students, scientists, regulatory agencies, funding agencies, journal editors and scientific societies. The published articles emphasize (1) Best practices in cell culture:  an overview, (2) Best practices for naming, receiving, and managing cells in culture, (3) Best practices for media selection for mammalian cells, (4) Best practices for the use and evaluation of animal serum as a component of cell culture medium; (5)  Best practices for authenticating cell lines, (6) Best practices for detecting and mitigating the risk of cell culture contaminants;  (7) Best practices for cryopreserving, thawing, recovering, and assessing cells; and (8) Best practices for storing and shipping cryopreserved cells.

Despite the fact that cells are fairly easy to manipulate and maintain, there is no consensus on how best to name, receive and manage cells into the laboratory (Reid YA, Best practices for naming, receiving, and managing cells in culture In Vitro Cell Dev Biol-Animal.  53(9):761-774. 2017).  Once a cell line is received into the laboratory, it is very important to have well-trained practitioners with the appropriate experience and expertise to ensure best practices in cell bank preparation, microscopic observation of cells in culture, cell growth optimization, cell enumeration, cell subcultivation and the use and naming of cells.  Moreover, the specialist should ensure that the appropriate certified facilities, equipment, validated reagents and supplies are available.

Yvonne A. Reid. Best Practices for naming, receiving, and managing cells in culture.  In Vitro Cellular & Developmental Biology-Animal, 53:761-774, 2017.


Best practices for media selection for mammalian cells

Paul J. Price

A cell culture medium is a complex mixture of nutrients and growth factors that, along with the physical environment, can either help or destroy your experiment or production run.  The classical cell culture media formulations consisted of amino acids, vitamins, inorganic salts and a carbon source for energy such as glucose.  The classical formulations require further supplementation with a protein source, such as animal sera and were designed for cancer-derived cell lines.  They have proven to be very sub-optimal for the growth of specialized cells, such as stem cells and differentiated cells. This review addresses the roles and problems associated with some of the key basic components of the cell culture medium and discusses formulation differences between serum-requiring, serum-free, xeno-free, animal-component-free, and chemically-defined media. The contribution of each component of the medium is essential for the maintenance of the cell type of interest. While some cell types, such as established human cancer cell lines, may be quite able to tolerate a range of culture conditions, many normal cells and stems cells are not. Optimization of each component and the physical parameters may be required to successfully maintain the latter cell types.  Emphasis is placed on optimizing cell growth and reducing apoptosis by optimizing osmolality and pH and controlling for ammonia, heavy metal toxicity and the production of free radicals. Nutritional requirements also differ between different  cell types  and functions and as cell  growth  proceeds,  different  cells  will  utilize  amino  acids  and  other components at different rates. By controlling for ammonia, free radicals, heavy metal toxicity, pH shifts, fluctuations in osmolality,   nutrient depletion, and chemical and biological contaminants, chances of success are significantly increased.  Methods for freezing and adapting cells to new formulations are also covered in this review.

Paul J. Price.  Best practices for media selection for mammalian cells.  In Vitro Cellular and Developmental Biology – Animal 53: 673-681, 2017


Best practices for the use and evaluation of animal serum as a component of cell culture medium

Dr. John Harbell (left) and Dr. Raymond Nims (right) are consultants advising clients in the biomedical sciences and pharmaceutical industries

In 2015, Dr. Yvonne Reid of the American Type Culture Collection assembled a small group of subject matter experts to write a series of Best Practices in Cell Culture papers for publication in the journal In Vitro Cell Dev Biol –Animal (see Baust et al. Best Practices in Cell Culture: an Overview. In Vitro Cell Dev Biol-Animal 53:669-672, 2017). Drs. Raymond Nims and John Harbell were given the assignment of addressing the best practices in the use of animal serum as a component of cell culture medium. The authors took on the task, however, based on the collective experience of the entire group of experts, both in the realms of cell culture itself as well as the ancillary topics of serum performance testing, storage and freeze-thaw impact, and adventitious agent risk and risk mitigation. Being users of serum as a component of medium for cell culture over the years, we have learned a variety of lessons the hard way and hoped to provide guidance that might save others the pain and expense associated with these lessons. In this regard, we believe the most important sections may be those discussing storage of serum, thawing of serum, and performance testing of serum. While reputable suppliers of serum will provide a certificate of analysis stating that the product is low in endotoxin and free of mycoplasma or viruses, there are aspects of serum quality that are out of the supplier’s control. These include the handling of the serum once in the user’s hands, and the performance requirements of the serum for the user’s specific culture system. The need to determine lot-to-lot variability for serum in the user’s cell culture system is another point that we thought needed emphasis. We ended with a frank discussion of the advantages and disadvantages associated with the use of animal serum as a medium component, and a short section on serum-free medium.

Raymond W. Nims and John W. Harbell. Best Practices for the use and evaluation of animal serum as a component of cell culture medium. In Vitro Cellular & Developmental Biology-Animal, 53:682-690, 2017.


Efficient protoplast isolation and transient gene expression system for Phalaenopsis hybrid cultivar ‘Ruili Beauty’

Members of the Laboratory of Ornamental Plant Biotechnology at Nanjing Agricultural University, from left to right: Chunling Tian, Wenting Mou, Yanxia Fu (front row), Song Liu, Guangdong Wang, Li Jiang, Yanna Li, Xiaoxiao Jiang, Jing Lao, Xingkai Tian.

 

Jinlan Li (The first author of the paper)

Xuezhu Liao

Shushan Zhou

 

Protoplast-based transient gene expression has wide applications on the gene functional analysis in plants. Although the method of protoplast isolation in Phalaenopsis has been reported, the isolation efficiency and quality need to be further improved. In this paper, an efficient protocol for protoplast isolation from mesophyll cells of in vitro plantlets of Phalaenopsis hybrid ‘Ruili Beauty’ was optimized by the single factor experiment and the orthogonal experiment. Furthermore, a stable and effective protocol for gene transient expression in Phalaenopsis protoplast via PEG-mediated method was established. Specifically, 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol with an enzymatic digestion time of 6 h was the optimal condition for protoplast isolation. This improved protocol could achieve yield as high as 5.94 × 106 (g FW)−1, which is 57.9% higher than the previous method (Shrestha et al. 2007). Furthermore, the effects of different factors on transformation efficiency of green fluorescent protein(GFP)gene were investigated and a stable system of transformation efficiency up to 41.7% was established with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 105 mL−1 protoplasts, and a transfection duration of 30 min, which is sufficient for subcellular localization studies or other investigations, such as promoter activity, protein-protein interactions and so on. This protocol will be of value for further gene functional analysis in Phalaenopsis, even for other orchids.

Jinlan Li, Xuezhu Liao, Shushan Zhou, Song Liu, Li Jiang, and Guangdong Wang. Efficient protoplast isolation and transient gene expression system for Phalaenopsis hybrid cultivar Ruili Beauty’. In Vitro Cellular & Developmental Biology-Plant, 54:87-93, 2018.