Textbook/lab manual for mammalian cell culture

Textbook/lab manual for mammalian cell culture

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For general techniques involving molecular biology, I know of Sambrook and Maniatis Molecular Cloning which covers many of the standard cloning techniques.

However, mammalian cell culture has many significant differences from bacterial cell culture, some of which include the many different variants of cell culture media as well as the relatively complex systems which cannot be provided using standardised media such as LB or TB in E. coli culture.

Are there any similar textbooks/lab manuals for cell culture? Alternatively, good review papers covering the different kinds of media used in mammalian cell culture and their relative benefits would also be a good answer to this question.

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications by Ian Freshney is the most popular book on the topic of animal cell culture.

You can also refer to ATCC animal cell culture guide which also has information about media formulations.

Cell Culture Engineering : Recombinant Protein Production

In Cell Culture Engineering: Recombinant Protein Production, editors Gyun Min Lee and Helene Faustrup Kildegaard assemble top class authors to present expert coverage of topics such as: cell line development for therapeutic protein production development of a transient gene expression upstream platform and CHO synthetic biology. They provide readers with everything they need to know about enhancing product and bioprocess attributes using genome-scale models of CHO metabolism omics data and mammalian systems biotechnology perfusion culture and much more.

This all-new, up-to-date reference covers all of the important aspects of cell culture engineering, including cell engineering, system biology approaches, and processing technology. It describes the challenges in cell line development and cell engineering, e.g. via gene editing tools like CRISPR/Cas9 and with the aim to engineer glycosylation patterns. Furthermore, it gives an overview about synthetic biology approaches applied to cell culture engineering and elaborates the use of CHO cells as common cell line for protein production. In addition, the book discusses the most important aspects of production processes, including cell culture media, batch, fed-batch, and perfusion processes as well as process analytical technology, quality by design, and scale down models.

-Covers key elements of cell culture engineering applied to the production of recombinant proteins for therapeutic use
-Focuses on mammalian and animal cells to help highlight synthetic and systems biology approaches to cell culture engineering, exemplified by the widely used CHO cell line
-Part of the renowned "Advanced Biotechnology" book series

Cell Culture Engineering: Recombinant Protein Production will appeal to biotechnologists, bioengineers, life scientists, chemical engineers, and PhD students in the life sciences.

Author Bios

Gyun Min Lee, PhD, is Professor at the Department of Biological Sciences at KAIST, South Korea, and heads the Animal Cell Engineering Laboratory. He is also Scientific Director at the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark.

Helene Faustrup Kildegaard, PhD, is a senior researcher and Co-PI for the CHO Cell Line Engineering and Design section at the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark (DTU).

Supplementary files

An integrated cell culture lab on a chip: modular microdevices for cultivation of mammalian cells and delivery into microfluidic microdroplets

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Chapter 48 : Cell Culture Bioreactors: Controls, Measurements, and Scale-Down Model

This chapter describes the types of bioreactors, bioreactor controls for process parameters, measurement of cell growth and metabolites, and a scale-down model using small- and pilot-scale culture vessels used in the biopharmaceutical industry. The major types of bioreactors used for cell culture processes in the biopharmaceutical industry today are stirred and airlift bioreactors. Recently, disposable bioreactors have been utilized for their convenience. Each type of bioreactor can be used for batch, fed-batch, and continuous cultures. Metabolites such as glucose, glutamine, lactate, ammonia, and amino acids can be monitored with an advantage of real-time measurements without sampling for in situ analysis of culture metabolites in order to maintain predetermined ranges of each metabolite in the fed-batch culture. The in situ probes will be widely used in the future due to the ease of real-time measurements and lack of sampling. Major changes in the metabolite levels are observed with lactate and ammonia, which are considered as by-products of culture, in addition to the changes in glucose, glutamine, and amino acid concentrations. The proper control of process parameters such as pH, dissolved oxygen (DO), agitation, and aeration is the most important consideration in the operation of bioreactors to consistently produce good cell growth, productivity, and quality of product. The scale-down model is also applied to the quality-by-design applications to develop a design space for process parameters to ensure predefined product quality.

Avoidance of Microbial Contamination

Potential sources of contamination include other cell lines, laboratory conditions and staff poorly trained in core areas such as aseptic techniques and good laboratory practice. Thus, the use of cells and reagents of known origin and quality alone is not sufficient to guarantee quality of product (cell stock or culture products) it is necessary to demonstrate quality throughout the production process and also in the final product. Routine screening aids the early detection of contamination since all manipulations are a potential source of contamination.

The three main types of microbial contaminants in tissue culture are:

Bacterial and Fungal Contamination

Bacterial contamination is generally visible to the naked eye and detected by a sudden increase in turbidity and colour change of the culture medium as the result of a change in pH. The cell culture may survive for a short time but the cells will eventually die. Daily microscopic observation of cultures will ensure early detection of contamination and enable appropriate action to be taken as soon as the first signs of contamination become apparent. In addition, specific tests for the detection of bacteria and fungi should be used as part of a routine and regular quality control screening procedure.

Mycoplasma Contamination

Mycoplasmas are the smallest free-living self-replicating prokaryotes. They lack a cell wall and the ability to synthesize one. They are 0.3μm in diameter and can be observed as filamentous or coccal forms. There are 6 major species that are tissue culture contaminants, namely M. hyorhinis, M. arginini, M. orale, M. fermentans, M.hominis and Acholeplasma laidlawii.

The effects of mycoplasma infection are more insidious than those of bacteria and fungi, inducing several long-term effects in cell cultures. These can include:

  • Altered growth rate
  • Morphological changes
  • Chromosome aberrations
  • Alterations in amino acid and nucleic acid metabolism

However, despite these well documented effects the presence of mycoplasma is often not tested for with the consequence that in such laboratories the majority of cell lines are positive for mycoplasma. Mycoplasma contamination is difficult to detect requiring the use of specialist techniques. In the past only specialist laboratories, such as culture collections, have performed these tests. However, a variety of commercial mycoplasma detection kits are now available although the performance characteristics of these kits can be extremely variable. A combination of these should be used as part of a routine and regular quality control screening procedure. ECACC tests cultures for the presence of mycoplasma on a routine basis and offers a mycoplasma testing service.

Some cell lines contain endogenous viruses and secrete virus particles or express viral antigens on their surface, e.g. Epstein-Barr Virus (EBV) transformed lines. These cell lines are not considered contaminated. However, bovine serum is a potential source of bovine viral diarrhoea virus (BVDV) contamination and use of infected serum will lead to contamination of cell lines with the virus. Contamination of cell lines with BVDV may cause slight changes in growth rate but since this virus is non-cytopathic, macroscopic and microscopic changes in the culture will not be detected. Suppliers of bovine serum are aware of this and screen sera accordingly generally, serum is sold as BVDV tested. Having said this you should be always check carefully to ensure that you understand the results of any testing that is performed on serum. Is the serum indicated as BVDV tested and none detected? What is the sensitivity of detection? BVDV tested serum is not necessarily BVDV free.

Introduction to cell culture

Cell culture is the growth of cells from an animal or plant in an artificial, controlled environment. Cells are removed either from the organism directly and disaggregated before cultivation or from a cell line or cell strain that has previously been established.

Certain culture conditions depend on the cell type, however, each culture must consist of a suitable vessel with a substrate or medium that supplies the nutrients (such as amino acids, carbohydrates, vitamins, minerals), growth factors or essential hormones for culturing cells. Gases (O2, CO2), physicochemical environment (pH, osmotic pressure, temperature) also play an important role to regulate the proper cell growth in an artificial environment.

Cell culture applications

Is a major consistent and reproducible tool in molecular and cellular biology.

Helps to study normal cell homeostasis, cell biochemistry, metabolism, mutagenesis, diseases, and compound effects.

Is a model system for diseases and drug screening.

Basic cell culture equipment

The specific equipment of a cell culture laboratory depends on the type of research conducted however, all cell culture laboratories have the same common goal: being free from pathogenic microorganisms.

The extended list below is corresponding to the equipment and supplies for the majority cell culture laboratories, that allows the work to be more efficient and accurate.

- 70% ethanol antiseptic - Cell incubator
- Aspiratory pump - Centrifuge
- Autoclave - Fridge/freezer
- Cell counter - Gloves
- Cell culture flasks - Media, sera, cell media additives
- Cell culture-grade Petri dishes - Pipettes of various capacities
- Cell culture-grade tubes of various sizes - Serological pipettor
- Cell culture hood - Sterile filters
- Cell culture microwell plates - Waste container

*Please note: The specific cell culture equipment depends on the cell type and aim of the study

Cell culture safety

Work in a cell culture laboratory is associated with different risk factors and hazards such as toxins or mutagenic reagents. The human/animal material may contain viruses and other dangerous biological agents. Therefore, while manipulating human/animal material it is important to follow general safety guidance for laboratory practices.

1. Wash hands when entering and before leaving the laboratory.

2. Wear safety clothes (gloves, closed shoes, lab coat).

3. No eating, drinking, smoking.

4. No or low aerosol creation.

5. Decontaminate all surfaces before and after the experiment.

6. Work in accordance with the facility guidelines.

7. Dispose of all waste in an appropriate way.

8. Restricted access to authorized personnel only.

9. Avoid using sharp objects.

10. Always clearly label all samples.

11. Report all incidents to the safety officer.

Aseptic techniques required while working with cell culture

To be successful in cell culture, it is essential to remain a contamination free environment (bacteria, fungi etc) Aspetic techniques ensure that no microorganisms enter the cell culture. Cell culture sterility is ensured by set of procedures.

Aseptic techniques required while working with cell culture.

Handling Reagents/Media Workplace
Slow/careful handling. Pre-sterilisation of all reagents/equipment Cell culture hood works properly
Sterilization of all items before starting. No contamination in reagents (expiration date, appearance normal). Frequent de-contamination (hood, fridge etc)
Sterile pipettes Work area: sterile and tidy
No touching of sterile items to non-sterilized surfaces

Cell culture environment

Cell culture is an amazing tool that allows for easy control and manipulation of all physiochemical and physiological cell factors, such as, temperature, osmotic pressure, pH, gas, hormones, and nutrients.

Cell culture environment.

Media pH Temperature CO2
Contains nutrients, growth factors, and hormones Average pH for mammatian cells is pH 7.4 Depends on body temperature of the host Controlled by media
Sera source of growth, tipids,hormones Mammalian cell lines 36-37°C Organic or C02 bicarbonate buffer systems are popular
Insert cell lines 27-30°C Can impact pH
4-10% C02 is most common

Media supplements for cell culture

Media supplements help to optimize cell growth for specific applications depending on the chosen tissue or cell type. The advantages of using media supplements such as growth factors or cytokines are that they may improve cell viability and growth and keep cells healthier for longer. For example, fibroblast growth factor (FGF) plays important roles in diverse biological functions in vivo and in vitro and can maintain cell culture over the weekend as Proteintech thermostable FGF (HZ-1285) does not require media changes every day.

The stability of FGFbasic-TS and FGF basic (E. coli-derived) in xeno-free, chemically defined cell culture media at 37˚C. The protein concentration was determined by ELISA each day for 3 days. After one day of incubation at 37˚C, FGF basic was undetectable, while FGFbasic-TS was present at levels of 60%, 35%, and 20% of its starting concentration at days 1, 2, and 3.

Growth factors play an essential role to maintain the in vitro culture growth. Depending on the environment, certain cells can give rise to a variety of lineage-specific cell types. See below a summary of the key growth factors needed for normal cell growth, metabolism, cell development in culture and their differentiation process.

Bone formation and regeneration.

Uses as differentiationfactor of pluripotent stem cellsand promotes osteogenic differentiation of mesenchymal stem cells

Acts as a mitogen for many cell types including hepatocytes

Promoting growth and differentiation of hematopoietic progenitor cells.

Used to support the in vitrocolony formation of granulocyte-macrophage progenitors.

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Sartoclear Dynamics ® Lab kits are designed for rapid harvesting 15 mL to 1,000 mL volumes of cell cultures in the lab, enabling clarification and sterile filtration to be performed in one step. These kits simplify the process by fully eliminating the centrifugation step otherwise needed for clarification. As a result, up to 1,000 mL cell cultures can be efficiently clarified and sterilized in minutes – quickly and easily.

Sartoclear Dynamics ® Lab Filtration Kits

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Clarification and sterile filtration of up to 1 L mammalian cell culture combining a filter aid for clarification and a vacuum filtration unit for sterile filtration.

  • Sterilized pouches of filter aid and vacuum filtration units
  • 14 kits to cover a range of sample volumes up to 1,000 mL with cell densities up to 20 x 10 6 cells/mL

Sartolab ® Multistation for Hands-Free Filtration of Small Volumes

For simultaneous filtration of up to 6 x 50 mL samples without the need for installation of extra connectors and time-consuming stabilization of filter units.

  • Hands-free filtration
  • One single vacuum source enables simultaneous filtration of up to 6 samples
  • Time-saving - no installation time for each filter unit before use

Sartoclear Dynamics ® Lab P15 Kit

Convenient, ready-to-use-kit combines a 20 mL syringe pre-filled with a filter aid for clarification with a 0.2 um polyethersulfone filter for sterile filtration.

The team at LifeArc, a medical research charity based in the UK, is using the Sartoclear Dynamics ® ️ Lab and Sartolab ® ️ Multistation to clarify high-density cell culture media in minutes, creating a workflow that’s more efficient and productive.

"Processing mammalian cell culture fluid can be particularly challenging, as in the lab we are often driven by the need to perform this task quickly with minimum loss in the quantity of cell culture fluid obtained. Standard vacuum filtration isn’t always the ideal solution due to limitations in the sophistication of the equipment itself. It often requires additional supervision and can be quite time-consuming to undertake, particularly when there are several samples involved.

The Multistation has significantly helped our lab. In addition to the six-sample capacity, the independently controlled multi-port design has provided operators with the flexibility to perform additional tasks in parallel, thereby delivering greater efficiency overall in the lab with improved cell culture fluid harvesting."

Textbook/lab manual for mammalian cell culture - Biology

Office: 208 Biosystems Research Complex
Clemson University
Clemson, SC 29634

Ph.D., University of Maryland, College Park, 1993,
Chemical Engineering

M.S., Colorado State University, Fort Collins, 1988,
Chemical Engineering

B.S.E., The University of Michigan, Ann Arbor, 1986,
Engineering Science - Bioengineering

Dr. Harcum completed her Bachelor of Science and Engineering degree from the University of Michigan in Ann Arbor in Engineering Science - Bioengineering. Her graduate studies were in Chemical Engineering she received her MS from Colorado State University and her PhD from the University of Maryland in College Park. She has worked for the Food and Drug Administration in the Center for Biologics Evaluation and Research. Specifically, she worked in the Division of Monoclonal Antibodies within the Office of Therapeutic Review and Research. She was a faculty member in the Department of Chemical Engineering and Molecular Biology Program at New Mexico State University from 1995-2002. She arrived at Clemson University in 2002 as an Associate Professor of Chemical Engineering. She transferred within Clemson to the Department of Bioengineering in 2004.

Dr. Harcum's research interests center primarily around recombinant DNA, bioreactor control, and gene expression. Her lab performs cutting edge research on a wide array of topics ranging from ethanol production from lignocellulosic biomass to protein aggregation in Chinese hamster ovary cells to large scale stem cell production to gene expression analysis of microorganisms. Dr. Harcum was invited to the National Academy of Engineering's Fifth Annual Symposium on Frontiers of Engineering, won an NSF CAREER Award, has been awarded the Center for Agricultural Biotechnology Fellowship and the Regent Fellowship, and was featured in the 1996-1997 edition of "Who's Who in Science and Engineering." Dr. Harcum and her collaborators in Electrical and Computer Engineering (Matthew Pepper, Li Wang, Ajay Padmakumar, Dr. Timothy C. Burg, and Dr. Richard E. Groff), won 1st Place in the BlueCompetition 2013 sponsored by BlueSens of Germany.

In collaboration with Johns Hopkins University, the University of Delaware, and the University of Massachusetts-Lowell, Dr. Harcum and Clemson University were awarded an NSF- Industrial/University Cooperative Research Center (I/UCRC) grant entitled the Advanced Mammalian Biomanufacturing Innovation Center (AMBIC). AMBIC is focused on reducing the time and cost of culturing the cells needed to make biopharmaceuticals. Additionally, Dr. Harcum is leading the team at Clemson as part of the newly form National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL). NIIMBL is led by the University of Delaware and is one of twelve Manufacturing USA Institutes. NIIMBL is focused on accelerating biopharmaceutical manufacturing innovation at all phases and educating and training a world-leading biopharmaceutical manufacturing workforce. Clemson researchers are very excited to be involved in this important initiative.

Textbook/lab manual for mammalian cell culture - Biology

The principal purpose of cell, tissue and organ culture is to isolate, at each level of organization, the parts from the whole organism for study in experimentally controlled environments. It is characteristic of intact organisms that a high degree of interrelationship exists and interaction occurs between the component parts. Cultivation in vitro places cells beyond the effects of the organism as a whole and of the products of all cells other than those introduced into the culture. Artificial environments may be designed to imitate the natural physiological one, or varied at will by the deliberate introduction of particular variables and stresses.

Virtually all types of cells or aggregates of cells may be studied in culture. Living cells can be examined by cinephotomicrography, and by direct, phase-contrast, interference, fluorescence, or ultraviolet microscopy. Fixed cells from culture are suitable for cytological, cytochemical, histological, histochemical, and electron microscopical study. Populations of cells from monolayers or suspension cultures are used for nutritional, biochemical, and immunological work.

Organ culture, the cultivation of whole organs or parts thereof, is particularly suitable for studies of development, of inductive interactions, and of the effects of chemical and physical agents upon the physiological functions of specific organs.

Both cell and organ culture have applications in pathology, e.g. , for comparative, developmental, and diagnostic studies of tissues from normal and diseased donors, for investigations on carcinogenesis, somatic cell genetic variation, viral susceptibility, etc. Cell cultures are widely used in microbiological studies, for investigations of the effects of radiation, and for screening drugs, especially carcinogenic, mutagenic, and radiomimetic agents.

Cell nutrition has been generally excluded from material selected for this chapter, since this topic has been fully reviewed elsewhere (Waymouth, 1954, 1960, 1965). The researchers to be referred to here have been chosen because they (1) contribute significantly to our understanding of the biology of the mouse, as distinguished from observations of general interest in cell biology ( e.g. , the use of mouse cells for testing or screening drugs or carcinogens), (2) refer to cells obtained from inbred strains of mice or from mutants, or (3) suggest application to such cells.

Tissues were first cultured before the turn of the century and since that time a multitude of techniques, each designed for the solution of a particular problem, has been devised. Basic information on technical procedures and their numerous applications in mammalian biology may be found in the textbooks of Parker ( 1961), Paul ( 1961), White ( 1963), and Merchant et. al. ( 1964). Other reviews include those edited by White ( 1957) and Stevenson ( 1962) a number particularly emphasizing cell nutrition by Stewart and Kirk ( 1954), Waymouth ( 1954, 1960, 1965), Hanks ( 1955), Biggers et al. ( 1957), Geyer ( 1958), Morgan ( 1958), Swim ( 1959), Paul ( 1960) and one dealing principally with cell biochemistry by Levintow and Eagle ( 1961). The Bibliography of the Research in Tissue Culture, 1884-1950 ( Murray and Kopech, 1953) covers the literature of that period very completely, and keys to papers in tissue culture for the years 1961 onwards are to be published (Murray and Kopech, 1965, 1966).

Although the chick embryo was the most generally used source of material for cultivation in the period 1910 to 1940, tissues of the embryonic and adult mouse have gained favor spectacularly since 1940, particularly since the introduction of antibiotics, which have reduced the need for strictly aseptic techniques in some types of experiment. The extensive cultivation of mouse cells owes much to the pioneering work of Earle, Evans, Sanford, and their colleagues at the National Cancer Institute. Among the major contributions of this group have been the improvement and standardization of techniques, including mass-culture methods and nutrition by chemically defined media, the development of cell lines and clones, the comparative study of the cytological and biochemical properties of long-established cell lines, and the investigation of malignant transformation in vitro .

Each of the basic cell types (fibrocytes or mechanocytes, epitheliocytes, and amebocytes (Willmer, 1960 a , 1960 b )) has its special characteristics in vitro . Experience has shown that it is important, when describing cells grown in vitro , to give details of the species, age, and sex of the source of the cells, the tissue of origin, and to state whether they are normal or neoplastic. Cells and tissues freshly isolated from the animal are designated "primary cultures" ( Fedoroff, 1966). A "primary cell line" refers to a population of cells derived by direct isolation from an animal and is not necessarily capable of serial propagation indefinitely. An established cell line refers to a population of cells which has been serially transplanted at least 60 time in vitro . Primary or established cell lines or cell strains should receive designations according to the principles recommended by the 1957 International Tissue Culture Meeting ( Anon., 1958 Paul, 1958 Fedoroff, 1966).

Applications of cell culture

Only a few representative examples of the use of cell cultures in mouse biology can be cited.

The mitotic process and its modification by stimulants or suppressors have been studied in many cell types ( Fell and Hughes, 1949). Exact chromosome counts, to establish the degree of divergence from diploidy, have been made on 10-day fetal mouse cells in culture by Hungerford ( 1955). The chromosome complements of newborn and adult mice can be counted, without killing the animals, in primary tissue cultures of tail tips or ear fragments ( Edwards, 1961). The mitotic cycle has been analyzed by Defendi and Manson ( 1963), actinomycin D-resistant and -sensitive systems or RNA synthesis identified by Paul and Struthers ( 1963), and the duration of the DNA synthetic period of mouse somatic cells shown to be probably constant ( Cameron, 1964).

Visible light (Earle, 1928 a , 1928 b , 1928 c Frédéric, 1954) has some inhibitory effects upon living cells. The lethal effects of X-irradiation can be quantified on mouse cells ( Reid and Gifford, 1952), and the effects of radiation upon cell constituents ( Whitmore et al. , 1958) and upon DNA and RNA synthesis ( Whitfield and Rixon, 1959 Till, 1961 a ) can be studied. Also methods of chemical protection of irradiated cells ( Whitfield et al. , 1962) can be applied. X-ray-induced chromosome aberrations can be analyzed ( Chu and Monesi, 1960). Ultraviolet light, which inhibits cell division is strain L cells, does not significantly affect DNA synthesis ( Whitfield et al. , 1961). The survival of irradiated cells can be compared in vivo and in vitro , as has been done by McCulloch and Till ( 1962) for mouse bone marrow exposed to Co 60 γ-rays.

Differentiation at the cellular level has mostly been studied in organ, rather than cell, cultures. However, Ginsburg ( 1963) has seen the differentiation of mouse thymus lymphoid cells into mast cells, which can be produced in large numbers and grown in suspension ( Ginsburg and Sachs, 1963). Muscle differentiation can also be followed in tissue culture, and the tissue culture technique has been applied by Pearce ( 1963) to the study of muscular dystrophy.

The uses of tissue culture in the study of cancer have been reviewed by Murray ( 1959). More recent work includes that of Mitsutani et al. ( 1960) on the development of a near-diploid cell strain from a smoke condensate-induced leiomyosarcoma in a male C3H mouse and that of Fernandes and Koprowska ( 1963) on cell lines from normal cervix uteri of C3H mice and on cells from uteri treated for varying lengths of time with benzpyrene.

Comparison of enzyme activities in cells in culture with those from the mouse have been made ( e.g. , of β-galactosidase by Maio and Rickenberg, 1960). Estimations of β-glucuronidase in cell lines from C3H mice (which have a lower activity in respect of this enzyme than most inbred mouse strains, especially in their livers) have demonstrated that most cell lines have activities many times higher than those of the highest activity mouse livers ( Kuff and Evans, 1961). On the other hand, mouse cell strains after long cultivation in vitro seem uniformly to have a very low catalase activity in comparison with freshly isolated mouse tissue ( Peppers et al. , 1960). Long-term cultures of fibroblasts seem to undergo greater variations in enzyme content than, of example, liver cells. Westfall et al. ( 1958) found that the arginase and rhodanese activities in liver cell lines were high, as in the tissue of origin, but that fibroblast lines varied widely in the activities of one or both of these enzymes. This variation may be related to Klein's ( 1960, 1961) experience that induction of arginase depends upon other factors than the substrate. In most cases arginase induction requires the presence of RNA as well as arginase. The enzyme patterns of established cell strains are among the most useful traits for characterizing cells in culture ( Westfall, 1962 Conklin et al. , 1962).

Stable and unstable characters of cell cultures

Cloning. In certain respects cells cultivated in vitro exhibit considerable stability in others they undergo extensive alterations from the parent cells. Cloning — the process of deriving a population of cells from a single cell — has enabled cell lines of common origin to be followed and has given many new insights into the potentialities of cells and into the circumstances under which they retain or lose characteristics derived from their parents ( Sanford et al. , 1948 Hobbs et al. , 1957 Sanford et al. , 1961 b ).

Variations within clones. The many cell lines and clones established from C3H mice by Sanford et al. ( 1961 e ) usually underwent morphological, biochemical, and chromosomal changes quite early in the culture history. But, once established, many of the characteristics were of remarkable stability and persisted over many years of serial cultivation. A clone of sarcoma-producing cells (originating from normal C3H connective tissue) gave rise to "high" and "low" sarcoma-producing lines ( Sanford et al. , 1954 Sanford et al. , 1958), and sublines of these were characterized by widely differing patterns of chromosome number and character ( Chu et al. , 1958), of several enzyme activities ( Sanford, 1958 Westfall et al. , 1958 Sanford et al. , 1959 Scott et al. , 1960 Peppers et al. , 1960 Sanford et al. , 1961 e ), and of glycolytic activity ( Woods et al. , 1959).

Stable characteristics of cell lines. Some functions persist over many transplant generations in culture. These include the production of melanin by a mouse melanoma ( Sanford et al. , 1952) and of steroids by an adrenal tumor ( Sato and Buonassisi, 1961) and the synthesis of 5-hydroxytyrptamine, histamine, and heparin by the mouse mast-cell tumor P-815 ( Dunn and Potter, 1957 Schindler et al. , 1959 Green and Day, 1960 Day and Green, 1962). The polynucleotide sequences of DNA characteristic of the mouse are retained in L cells after more than 20 years in vitro in spite of the very abnormal karyotype exhibited ( McCarthy and Hoyer, 1964).

Immunological characteristics. Immunological specificity persists over very long periods of cultivation in homologous, heterologous, or chemically defined media, particularly mouse-strain specificity in tumors cultivated in vitro . Immunological methods have been used for species identification of cells in culture ( Coombs et al. , 1961 Coombs, 1962 Fedoroff, 1962 Brand and Syverton, 1962), for studying antigenic differences between cell lines ( Coriell et al. , 1958 Kite and Merchant, 1961 McKenna and Blakemore, 1962), and for identification of particular antigens, such as the H-2 transplantation antigen (Manson et al. , 1962 a , 1962 b Cann and Herzenberg, 1963 a , 1963 b ).

Neoplastic transformations. It has been repeatedly observed that cells of normal origin undergo malignant change after more or less prolonged cultivation in vitro ( Earle and Nettleship, 1943 Sanford et al. , 1950 Evans et al. , 1958 Sanford et al. , 1961 d , 1961 e ). Neoplastic transformation of cells originating from normal tissues and the maintenance or loss of the capacity to produce tumors on transplantation into suitable hosts have been intriguing problems illustrating the range of capabilities of the cell. Many of these transformations have been "spontaneous" ( i.e. , unexplained) ( Earle and Nettleship, 1943 Sanford, 1958, 1962 Evans et al. , 1958 Sanford et al. , 1959 Shelton et al. , 1963). Others have been deliberately induced by chemical carcinogens ( Earle, 1943 Earle et al. , 1950 Sanford et al. , 1950 Shelton and Earl, 1951 Berwald and Sachs, 1963) or by viruses ( Dawe and Law, 1959 Dulbecco and Vogt, 1960 Vogt and Dulbecco, 1960 Sanford et al. , 1961 c Sachs and Medina, 1961 Pearson, 1962).

Tumors retain their capacity to grow in histocompatible hosts and in general do not acquire the ability to transcend histocompatibility barriers, except after being stored at -70°C ( Morgan et al. , 1956), though some degree of immunological incompatibility can develop in long-term cultures (Sanford et al. , 1954, 1956). Strains of cells, originating from normal cells and acquiring within one or two years the ability to produce tumors, may undergo a progressive reduction in tumor-producing capacity after several more years in vitro ( Earle et al. , 1950). The cells with reduced tumor-producing ability could, however, grow in animals which had received X-irradiation ( Sanford et al. , 1956). The strain L, for example, after 10 year in vitro could produce tumors in 15 per cent of unirradiated and in 64 per cent of irradiated C3H hosts. This raises the question whether to progressive immunological incompatibility is due to changes in the cell line or to changes in the inbred strain over the period of 10 or more years since the cell isolation. After 13 years the L cells retained their C3H specificity, i.e. , would not grow in any of several other strains of mice. The differences in the "high" and "low" cancer-producing lines, both of which produce some immunity in C3H mice, and the fact that the "low" line will grow in irradiated C3H hosts, led to the conclusion ( Sanford et al. , 1958) that the faster-growing "high" tumor line can establish a tumor before resistance develops in the host. Cell strains and derived single-cell clones, established in culture from C3H carcinomas carrying mammary tumor agent, were found to vary in the persistence of the agent. In some cell strains the agent was demonstrable after 6 to 12 months of rapid cell proliferation in vitro in others the agent disappeared ( Sanford et al. , 1961 a ).

Experimental control of malignant change. Attempts have been made to place the "spontaneous" transformation of normal to malignant cells under experimental control. Evans et al. ( 1964) have followed the progressive changes in cultures initiated from minced C3H embryos by testing their ability to produce tumors on intraocular implantation. No tumors resulted from a limited number of cultures grown for up to 211 days in a chemically defined medium. Cells grown in a medium supplemented with 10 per cent horse serum were able to produce tumors from about 120 days.

Barski and Cassigena ( 1963) aimed to produce parallel malignant and nonmalignant cell lines from adult female C57BL lung, analogous to the spontaneously derived parallel C3H lines of Sanford et al. ( 1950), for use in their studies of cell hybridization (see below). Such pairs of lines were derived, one from a culture frequently subcultured with the aid of trypsin, the other from a less frequently transferred culture, subcultured by mechanical dispersion and not exposed to trypsin. Both lines early became aneuploid (mean chromosome number in both cell lines at the 10th passage of the trypsinized line and the sixth passage of the nontrypsinized line was 68). The trypsinized line (PT) was highly malignant from the 16th passage (184 days), whereas the nontrypsinized line (PG) was not malignant up to the 37th passage (436 days). Further evidence is needed to determine whether the differences in morphology and malignancy are causatively related to the trypsin treatment. Todaro and Green ( 1963) suggested that the process of establishment of cell lines may require a reduction in the "leakiness" of cells to small molecules, and trypsin treatment increases the "leakiness" ( Phillips and Terryberry, 1957 Magee et al. , 1958).

Sarcomatous change in carcinomas. Tissue cultures have contributed to the elucidation of the well-known "sarcomatous change" frequently observed in transplanted carcinomas. In an extensive study Sanford et al. ( 1961 d ) examined 18 different cell strains derived from C3H mammary gland tumors. In general, tumors maintained in culture for up to 25 weeks grew as differentiated mammary carcinomas on retransplantation into mice. Cells transplanted after this time grew as sarcomalike tumors. It appeared that tumors which had been carried in mouse serial passage were morphologically more stable than primary tumors put into culture. The "sarcomatous change" was apparently not a unitary process. In some instances, with hepatomas, melanomas, and thyroid tumors ( Sanford et al. , 1952), as well as with mammary tumors, the stroma may undergo malignant change. In other cases, the carcinoma cells themselves change morphologically and assume a fibroblastic appearance. The opposite change (from fibroblastic to epithelioid cell types) has been studied in malignant origin by Ludovici et al. ( 1962 a , 1962 b ), who produced a significantly higher (64 per cent) proportion of cultures showing this alteration by treatment with a trypsin-antibiotic mixture, than was seen in controls not so treated (7 per cent).

Chromosomal variation and somatic cell genetics. Nutrient-dependent and nutrient-independent, drug-resistant and drug-sensitive, radiation-resistant and radiation-sensitive clones ( Hauschka, 1957 Fedoroff and Cook, 1959 Fisher, 1959 Hsu and Kellogg, 1959 Biesele et al. , 1959 Roosa and Herzenberg, 1959 Hsu, 1961 Cann and Herzenberg, 1963 a , 1963 b ) are among the tools making possible the study of the genetics of mammalian cell and neoplastic cell population ( Merchant and Neel, 1962 Harris, 1964 Krooth, 1964).

Rothfels and Parker ( 1959) reported, what is now a rather common experience, that freshly explanted tissues (in their case from CF 1 mice) grow rapidly at first, then pass into a long (6 months or more) period of survival without growth, and finally, in some instances, enter a new phase of proliferation from which cell lines may be established. The chromosomes of such cell lines are usually heteroploid and heterotypic ( i.e. , contain chromosomes differing markedly from the normal 40 telocentrics of the mouse). Bimodal chromosome distributions are not uncommon, as in the case of Rothfels and Parker's culture 23855-8 from CF 1 kidney, which contained approximately equal numbers of cells with around 38 and 70 chromosomes respectively, a pattern which persisted through at least 14 subcultures (12 months). Hsu ( 1961) and Chu ( 1962) commented upon the rapid departure from diploidy observed even in primary cultures with mouse cells and contrast this with the greater karyotypic stability of man, rat, and many other mammals. Todaro and Green ( 1963) developed established cell lines from trypsin-disaggregated 17- to 19-day mouse embryos using trypsin at each transfer. The usual decline in growth rate during early passages was encountered but, at from 15 to 30 generations in vitro , the growth rate rose. At the beginning of the third phase which, under their conditions, was less than 3 months, the cells responsible for the upturn in growth rate were diploid, but they shifted (often rapidly) to the tetraploid range. Marker chromosomes appeared later.

Long-established cell strains, like most cancers (which Hauschka ( 1958) describes as "multiclonal mosaics of altered karyotypes"), have characteristic and identifiable karyotypes, even though within a strain there may be considerable variation of numbers and types of chromosomes. It is rare to find in cultures of mouse cells that the chromosomes are all, or even sometimes predominant, telocentric. However, an analysis by Levan and Hsu ( 1960) of NCTC 2940 — a cell line which originated from a C3H mammary carcinoma — after being carried for about 2 years in vitro , showed only telocentrics in a stem line number of s = 84 chromosomes. Another mammary tumor cell line, NCTC 2777, of hypertetraploid number ( s = 73), contained only telocentrics, with the marked exception of one large, bizarre, and multiform heterometacentric chromosome.

Five established strains of mouse cell (three of them sublines of NCTC clone 929, strain L) were found by Hsu and Klatt ( 1958) to exhibit karyotypic polymorphism and to contain "marker" chromosomes highly characteristic of individual cell strains and wholly distinct from normal mouse chromosomes. In another study Hsu ( 1959) observed modal chromosome numbers of 67 to 73 in 12 mouse cell strains. Highly polyploid cells are not uncommon. Levan and Biesele ( 1958) observed a gradual increase in the number of polyploid cells in cultures of mouse embryo cells. Polyploid cells can usually be found early in the life of cultures, and their proportion in the population can be increased by treatment with colchicine ( Hsu and Kellogg, 1960). One subline (L-P59) of NCTC clone 929 strain L, studied by Hsu ( 1960), contained 63 to 65 chromosomes, including a very conspicuous long subtelocentric (chromosome D). Derived subline Amy from L-P59 had an average chromosome number of 128 (with two D chromosomes), and subline Barbara had 58 to 59 without the D marker but with a large metacentric chromosome known as Victoria. In mixed cultures the stem line L-P59 rapidly overgrew Amy or Barbara. Moreover, the proportion of D chromosomes in L-P59 cultures was found to be variable according to the frequency of subculture. Old cultures, or cultures subdivided only every 2 weeks, contained on an average more than 1.5 D chromosomes per cell. In cultures subdivided twice a week, the population changed to one with less than one D chromosome per cell. The D chromosome and a probable isochromosome T were lost from one subline (L-M) ( Hsu and Merchant, 1961) and new distinctive markers were reported 2 years after the first study. Hsu ( 1961) has reviewed the topic of chromosomal evolution in cell populations.

Occasionally mouse cell lines of diploid mode have been observed. Billen and Debrunner ( 1960) had cells from normal mouse bone marrow which remained diploid for more than 1 year. A line (H 2 ), started from cells from the peritoneum of a C3H mouse, retained its diploid character for at least 5 months before becoming predominantly tetraploid with a minority of hypodiploid (38) cells, which gradually diminished ( Hsu et al. , 1961). Another series of intriguing hypodiploid cell lines are the MB III lymphoblasts which originated in 1935 from a spontaneous lymphosarcoma T86157 in a 286-day-old female mouse ( De Bruyn et al. , 1949). The primary line (MB I) of lymphosarcoma cells contained a mixed population of tumor-producing lymphoblasts ( s = 40 or 41) and tumor-negative fibroblasts ( s = 56). The MB III lines are sublines of lymphoblasts, free from fibroblasts, which have become tumor-negative and hypodiploid ( s = 30 to 32). In contrast to many normal cells which undergo "spontaneous" transformation in vitro into tumor-producing cells, neither MB III (lymphoblasts) nor MB II (fibroblasts), in spite of great morphological variability and frequent mitotic disturbances, produces tumors in vivo after about 27 years of life in vitro ( De Bruyn and Hansen-Melander, 1962).

The radiosensitivity of sublines of strain L mouse cells, as measured by their ability to form macroscopic colonies, was found to be independent of chromosome number in cell lines with mean chromosome numbers between 53 and 109 ( Till, 1961 b ). Chromosomal anomalies acquired during in vivo , by injecting a teratogen during pregnancy, persisted in the fetal tissues during cell culture, the treated cells showing 50 per cent polyploidy, and the controls only 2 per cent ( Ingalls et al. , 1963).

Cell hybridization. By making cultures of populations of two cell types, each containing conspicuous marker chromosomes, cells can be produced containing both sets of chromosomes. Sorieul and Ephrussi ( 1961), Barski ( 1961), and Barski et al. ( 1961 a ) found such "hybrid" cells in mixed cultures of cells from NCTC 2472 (a high-cancer line) and NCTC 2555 (a low-cancer line). Both of these lines took their ultimate origin from a single clone of normal cells, which produced the original "high" and "low" lines ( Sanford et al. , 1954) later designated NCTC 1742 and NCTC 2049 respectively. NCTC 2472 was derived from NCTC 1742 ( Sanford et al. , 1961 e ) and NCTC 2555 from NCTC 2049 ( Woods et al. , 1959). The high-cancer line NCTC 2472 (N1) has a modal chromosome number of 55 telocentrics, one being very long (Barski et al. , 1961 b ). The low-cancer line NCTC 2555 (N2) has a modal chromosome number of 62, with from 9 to 19 two-armed chromosomes. By 104 days in mixed culture M type cells began to appear, with 115 to 116 chromosomes, of which 9 to 15 were metacentric, and in which the extra-long telocentric chromosome could usually be identified. Cells of the M type were never found in cultures of N1 or N2 cells alone ( Barski and Cornefert, 1962) but M cells could be produced in vivo as well as in vitro , in tumors produced by inoculating C3H mice with mixed-cell populations. The hybrid characteristics of cloned M lines remained stable for at least 1 year. Barski and Belehradek ( 1963) have demonstrated cinephotomicrographically that nuclear transfer can take place in mixed cultures of N1 cells with normal mouse embryo cells. This may occur repeatedly in mixed cultures, or, as Ephrussi and Sorieul ( 1962) point out, it is not impossible that hybrid populations "arose from a single mating event involving modal cells of the two parental lines, followed by rapid segregation."

Techniques for culturing organs are described in the textbooks of Parker ( 1961), Paul ( 1961), White ( 1963), and Merchant et al. ( 1964), and are referred to in the major papers and review articles of the principal practitioners of the method, e.g. , Wolff ( 1952), Fell ( 1953, 1954, 1955, 1958, 1964), Gaillard ( 1942, 1948, 1953), Borghese ( 1958), Kahn ( 1958), Lasnitzki ( 1958, 1965), Trowell ( 1959, 1961 b ), and Grobstein ( 1962).

Organ culture is used principally for (1) the maintenance of structural organization in tissues which are to be subjected to experimentally varied environments ( e.g. , to hormones, drugs, or radiation) (2) the study of morphogenesis, differentiation, and function in excised organs or presumptive organs and (3) for comparison of the growth and behavior of explanted organs with the growth and behavior of similar organs in sit .

Almost every organ of the mouse has been cultivated in vitro . Some of the principal references to the cultivation of mouse organs are listed in Table 25-1.

The environmental variables studied by means of organ cultures include: radiation ( Trowell, 1961 a Lasnitzki, 1961 a , 1961 c , 1961 d Borghese, 1961), vitamins, mainly vitamin A ( Fell and Mellanby, 1952, Lasnitzki 1958, 1961 b , 1961 c , 1962 New 1962) and carcinogens ( Lasnitzki, 1958 Lasnitzki and Lucy, 1961). Organ culture is peculiarly suitable for the study of hormones ( Fell, 1964) and provides an excellent way of distinguishing the effects of individual, or combinations of several, hormones on particular structures ( Table 25-2).

The mammary gland has been one of the most commonly grown organs of the mouse and, besides its use for investigation of responses to hormones, has been studied for its secretory activity ( Lasfargues, 1957 b Lasfargues and Feldman, 1963) and as a vehicle for the mammary tumor agent ( Lasfargues et al. , 1958). The toxic effects of steroid hormones on mammary adenocarcinomas of C3H mice in organ culture have been examined ( Rivera et al. , 1963).

The cultivation of mouse ova (Whitten, 1956, 1957 Tarkowski, 1959 a , 1959 b ) has made possible the production in vitro of genotypically mosaic embryos from fused eggs (Tarkowski, 1961, 1963 Mintz, 1962 c , 1963).

Organ culture has contributed significantly to our understanding of embryonic induction and of the control of morphogenesis by the juxtaposition of specific cell types. The effects of specific mesenchymal elements upon epithelial structures has been elucidated, e.g. , by Borghese ( 1950 a , 1950 b ), Grobstein ( 1953 a , 1953 b , 1953 c , 1955 a , 1955 b , 1956, 1957, 1959, 1962), Grobstein and Dalton ( 1957), Auerbach and Grobstein ( 1958), and Auerbach ( 1960, 1961 a , 1961 b ). Cartilage induction has been studied in BALB/c x C3H embryos by Grobstein and Parker ( 1954) and Grobstein and Holtzer ( 1955) and in T / T embryos by Bennett ( 1958).

The pioneer work of Hardy ( 1949, 1951) in the growth of hair and hair follicles has been followed up by Cleffman ( 1963) with studies of pigment formation in the hair-follicle melanocytes of agouti mice.

The mouse has become one of the species of choice for furnishing tissue for cell and organ cultures. A fund of basic information on mouse tissue culture is growing, much of it concerning work with tissues from inbred strains of mice. In so far as the genetic history of the cells and organs cultivated, as well as their subsequent history under cultivation, is pertinent to their observed behavior, this information will be valuable for future work on the characterization, function, and variation of somatic cells.

1 The writing of this chapter was supported in part by a grant from The John A. Hartford Foundation.

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How can I do base editing in my lab?

Here is a step-by-step workflow that you can use to carry out base editing in mammalian cells via plasmid-based delivery methods:

  1. Design sgRNAs with the web-tool we developed with Benchling that incorporates these rules.
  2. Order oligos and clone your sgRNA expression plasmid.
  3. Prepare transfection-quality plasmids for your sgRNA plasmid and base editor of choice.  We recommend BE3 for most applications. If low amounts of indel formation are not acceptable, then we recommend using BE2. The plasmids can be obtained from Addgene.
  4. Transfect or nucleofect your cells of interest with the sgRNA and BE plasmids. In general, a 1:3 ratio of sgRNA plasmid:BE plasmid (by weight) has been found to give the best results.
  5. After 3 days, harvest transfected cells and quantify your base editing efficiency using HTS.

For a full explanation of how we determined these rules, please see our full manuscript in Nature.