Cell Culture Techniques

NOTE: Not applicable to Clonetics® and Poietics® Primary Human or Animal Cells.

If you are looking for more details, kindly visit cell culture factories system.

In cell culture there is frequently the need to subculture cells. In doing so, cells can be propagated for the purposes of increasing cell numbers or providing cells in a culture vessel suitable to one’s needs. There are a number of ways to remove cells from one culture vessel and pass them to another vessel.

 

Cells may be removed from surfaces on which they are attached by:

• Mechanical means (scraping)
• Chelating agents, ethylenediaminetetraacetic acid (EDTA)
• Enzymes (trypsin, pronase, collagenase)

 

Enzymes and chelating agents are often used in combination. Trypsin is an aqueous crude extract prepared from porcine pancreas. It is the most common means used for removal of cells from surfaces and from intact tissue. Trypsin is, to some extent, a misnomer because in addition to trypsin, the preparation contains other proteases, lipases, and carbohydrases. The multitude of digestive enzymes produced by the pancreas would be expected to be found in trypsin preparations. Pure crystalline trypsin can be used, but it is more expensive than crude trypsin and often does not work as well, especially when preparing cells from intact tissue.

The optimum conditions for trypsin activity are a pH range of 7.6–7.8 and a temperature of 37°C. The effect of trypsin is to break down the intracellular matrix that binds cells to each other or to a substrate surface. There are no chemical standards for trypsin activity. We conduct quality assurance tests on trypsin to determine its capacity to detach cells from a substrate surface in a standard time period without damage. This is in addition to the usual tests for sterility.

Trypsin is typically used at concentrations between 0.05% and 0.25%, although some applications may require concentrations outside this range. Versene® Solution (EDTA) enhances trypsin action, and therefore lowers the required trypsin concentration for effective performance. Concentrated trypsin (2.5%) should be diluted in calcium- and magnesium-free balanced salt solution (BSS) or Dulbecco’s Phosphate Buffered Saline. Dilution in water is not recommended since the solution will be hypotonic and produce cell damage. Dilution in saline alone is also damaging to cells.

 

Trypsinization Procedure

Cell cultures are normally subcultured (“split”) when the cultures are at or near confluency. As a general rule, the longer the time frame between when confluency is first achieved and subculturing, the longer it will take for the trypsin to act.

 

1. Decant medium from the culture vessel. Serum inhibits trypsin activity, so complete removal of serumcontaining medium is necessary.

 

2. Rinse the cell sheet with BSS without calcium and magnesium before addition of Trypsin/Versene®. The monolayer should be thoroughly covered with BSS. This rinse is instantaneous but the BSS can remain on the cell sheet for up to 4 hours, if desired.

 

3. Pour off rinse medium. Trypsin/Versene® is to be added to each vessel as follows:
75 cm2 → 2.5 mL to 5.0 mL
150 cm2 → 5.0 mL to 10.0 mL
850 cm2 roller bottle → 10.0 mL to 20.0 mL

 

4. Cover the monolayer thoroughly with Trypsin/Versene®. Since different lots of Trypsin/Versene® may vary in strength, it is acceptable to monitor the trypsinization process at room temperature for the first 30 seconds.This will ensure that the trypsinization process is not occurring too rapidly.

 

5. The culture vessel should then be moderately hit against the palm of the hand to see if the cells are being dislodged. Hold the vessel up to a light in a vertical position and look for signs of the cell sheet sloughing off of the surface. If the entire monolayer is dislodged, proceed to step #6. If not, incubate at 37°C and observe the vessel every minute for dissociation. The culture vessel should again be hit against the palm of the hand to ensure all cells have been dislodged. Remove culture vessel from the incubator.

 

6. Immediately transfer dissociated cells to a vessel containing medium supplemented with 10% serum. All of the cells should be removed. Aspirate the medium plus cells with a pipette onto the surface to remove all remaining cells. It is essential that this aspiration be done as completely as possible with a small bore pipette so as to obtain individual, dispersed cells. If the cells are not separated, the new culture will contain numerous microcolonies. Cells added to the vessel should be stirred with a magnetic stir bar at a speed that avoids vortexing (approximately 100–200 rpm), or agitated frequently. It is important at this point to add medium containing serum at least 10 times the volume of Trypsin/Versene® used. This will ensure that the digestive agent is inhibited.

 

7. Add suffcient fresh medium to the aspirated suspension so that the total volume will cover the surface of two culture vessels, each having the same surface area as the original culture vessel (or use a single culture vessel having twice the floor area of the original vessel). This is a 1:2 split. Other split ratios can be used for vigorously growing cell populations.

 

8. Incubate the culture vessel (or vessels) at 37°C.

 

9. When making 1:2 splits, subculturing of human diploid cell cultures should be done on a rigid 3 or 4 day schedule, at which time confluent sheets should occur. Surplus cells can be frozen and stored in liquid nitrogen.

 

10. Populations that can be cultivated indefnitely can be subcultured serially each time confluency is reached. If the culture is a diploid population with a finite doubling capacity, increase the population doubling level (PDL) number by one at each 1:2 subculturing (split).

 

11. By making repeated 1:2 splits (twice a week) it can be seen that the number of culture vessels can be built up geo metrically (1, 2, 4, 8, 16, 32, 64, etc.) in a short period of time for the production of large quantities of cells for various purposes.

 

12. Although the line will be eventually lost as a continuously passaged line, it will not be lost for use since frozen ampoules can be obtained at almost every passage and thus the line can be restored to continuous passage again, up to a cumulative total of about 50 population doublings. By repeating this procedure, the number of cells that can be obtained is almost unlimited for all practical purposes.

 

13. A human embryonic diploid line has an in vitro life span of about fifty 1:2 subcultivations, or population doublings, at which time the cells will cease to divide and eventually die.

 

14. Using split ratios higher than 1:2 results in the advantage of minimizing the number of manipulations necessary to obtain a specific cell density or number of culture vessels. Since human embryonic diploid cell lines pass through a finite number of population doublings in vitro, it is necessary to keep a record of the number of population doublings that have elapsed. With a 1:2 split ratio this is achieved by simply adding “1” to each split since this ratio yields one population doubling. Larger split ratios can be used. For example, a split ratio of 1:4 would yield 2 doublings per 1:4 split; a 1:8 split ratio would yield 3 doublings per 1:8 split. In order to have knowledge of the approach of cessation, it is essential to keep records of the number of elapsed population doublings.

 

15. Since human diploid cells multiply by fission, the increase in population may be expressed per cell as follows:

 

Number of Cells 1  2 4 8 16 Population Doubling Level 0 1 2 3 4

 

References

1. Hayflick, L. and Moorhead, P.S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Research 25:585.
2. Hayflick, L. (1970) Aging under glass. Exp. Geront. 5:291.
3. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37:614. 
4. Hayflick, L. (1968) Human cells and aging. Scientific American 218:32.
5. Hayflick, L. (1973) Subculturing human diploid _broblast cultures. Methods and Applications of Tissue Culture Eds. Patterson, M.K. and Kruse, P.F., Academic Press, N.Y.
6. Freshney, R.I. (1983) Culture of Animal Cells: A Manual of Basic Technique. Alan R. Liss, Inc., New York

 

Associated Data

Data Availability Statement

More experimental details about original data depicted in , and will be made available by the corresponding author upon reasonable request.

Abstract

The cultivation of cells in a favorable artificial environment has become a versatile tool in cellular and molecular biology. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research. However, despite their important role, cell lines are frequently misidentified or contaminated by other cells, bacteria, fungi, yeast, viruses, or chemicals. In addition, handling and manipulating of cells is associated with specific biological and chemical hazards requiring special safeguards such as biosafety cabinets, enclosed containers, and other specialized protective equipment to minimize the risk of exposure to hazardous materials and to guarantee aseptic work conditions. This review provides a brief introduction about the most common problems encountered in cell culture laboratories and some guidelines on preventing or tackling respective problems.

Keywords:

contamination, mycoplasma, retrovirus, STR profiling, misidentification, cell authentication, conditional reprogramming

1. Introduction

Cell culture experiments are widely used in biomedical research, regenerative medicine, and biotechnological production. Due to restrictions on the use of laboratory animals by animal protection laws and the strict implementation of the 3Rs (Replacement, Reduction, and Refinement) formulated by William Russell and Rex Burch to improve the welfare of animals, it can be expected that the general use of cell lines will further increase during the next years to substitute animal-based research [1]. However, it should be noted that cell culture experiments, when not properly conducted, are prone to errors. Therefore, it is essential that cell culture studies are performed with good cell culture practice (GCCP) to assure the reproducibility of in vitro experimentation [2].

For more t 25 flaskinformation, please contact us. We will provide professional answers.

Explore more:
Body & Hand Lotion Bottle Packaging - Cosmopacks

In particular, inter- and intra-specific cross-contamination and cell misidentification, genetic drift, contamination with bacteria, fungi, yeast, viruses, or chemicals, and lack of quality control testing are widespread fatal cell culture problems that contaminate the literature with false and irreproducible results [3]. Rough estimates suggest that the number of published papers that used problematic cell lines is about 16.1% [4]. Moreover, the International Cell Line Authentication Committee (ICLAC) lists 576 misidentified or cross-contaminated cell lines in its latest register released in June 2021 [5].

Although it is hard to estimate how much misguided articles are actually affected, there is still an urgent need to better sensitize scientists to this problem. Furthermore, biosafety and ethical aspects are not in the public awareness or are even ignored when working with cell lines. Exemplarily, several continuous growing cell lines were established by transformation with the Simian virus 40 (SV40) large T-antigen (SV40T) or other agents with oncogenic potential, immortalized by introducing telomerase reverse transcriptase (TERT) activity, derived from genetically modified animals, or by novel technologies such as CRISPR/Cas9 gene editing [6,7,8]. Consequently, many cell lines need to be classified as genetically modified cell lines (GMCLs) that need sufficient safety attention.

During the last decades, scientists have established many guidelines on good cell and tissue culture practice (GCCP) that provide continuously updated guidance on the main principles to consider when performing cell culture. The GCCP guidelines highlight issues of quality management, background on culture systems, documentation and reporting, general safety instructions, information about education and training, and ethical issues associated with the performance of cell culture experiments [2,9]. Moreover, these expert documents try to promote the harmonization, rationalization, and standardization of laboratory practices including manufacture and testing to foster the compliance of researchers’ work with laws, regulations, and ethical principles. In addition, these guidelines provide extensive information about essential, beneficial, and useful additional equipment for setting up and furnishing a cell culture laboratory environment with a focus on national and international agreed standards. These guidelines are rather complex and comprehensive, so they will be of interest primarily to trained researchers, who have extensive experience in performing cell culture experiments for many years.

In this article, some general aspects of working with cell lines are discussed on a more simplified level. In particular, tests for the authentication of cell lines, potential cell culture contaminants, and brief information about ethical issues and biological safety guidelines for use of cell lines in biomedical research are summarized. As such, the information provided should be useful for those that will start to conduct cell culture experiments.

2. Classification of Cell Culture Types

Cell lines can be roughly classified into three groups, namely (i) finite cell lines, (ii) continuous cell lines, also known as immortalized or indefinite cell lines, and (iii) stem cell lines [2]. Finite cell lines are normally derived from primary cultures and have slow growth rates. As such, they can be grown for a limited number of cell generations in culture before finally undergoing aging and senescence, a process that is indicated by loss of the typical cell shape and enrichment of cytoplasmic lipids. Importantly, finite cell lines are contact-inhibited and arrested in the G0, G1, or G2 phase after forming monolayers [10].

In contrast, continuous growing cell lines are typically obtained from transformed or cancerous cells and divide rapidly and achieve much higher cell densities in culture than finite cell lines. In some cases, these cell lines exhibit aneuploidy (i.e., one or more chromosomes being present in greater or lesser number than the others) or heteroploidy (i.e., having a chromosome number that is other than a simple multiple of the haploid number). They often can be grown under reduced serum concentrations, are not contact-inhibited, and might form multilayers. Stem cells are an undifferentiated or partially differentiated pluripotent cell type originating from a multicellular organism. These cells can be extended to indefinitely more cells of the same type or alternatively can be triggered under the right conditions to produce cells with specialized functions. As such, they can act as a kind of multipotent precursor for many different cell types.

In all cases, the growth of cells from various sources requires an artificial but controlled environment, in which sometimes highly specialized media, supplements, and growth factors are needed for proper cell growth. A cell type can either grow adherent (attached to a surface) requiring a detaching agent for passaging, or alternatively can be free floating in suspension. Adherent cells can be further divided into fibroblast-like cells having an elongated shape and epithelial-like cells characterized by a polygonal shape. Similarly, each cell culture can have unique properties in regard to morphology, viability, doubling time, and genetic stability and their handling and maintenance may require different media, culture conditions, and additives or processing agents including antibiotics, detachment solutions, or surface coating for cell attachment [2]. Non-adherent or suspension cells grow either as single cells or as free-floating clumps in liquid medium that do not require enzymatic or mechanical dissociation during passaging. However, in some cases, these cells demand shaking or stirring for adequate gas exchange and proper growth. Typical examples of non-adherent cells are hematopoietic cell lines derived from blood, spleen, or bone marrow that proliferate without being attached to a substratum. Nevertheless, also some adherent cell lines can be adapted to grow in suspension, which allows for more manageable cell culturing at larger scales with higher yields in special applications [11,12]. In addition, compared to adherent cells, cells grown in suspension are generally easier to handle. Exemplarily, when adherent cells should be analyzed by analytical flow cytometry or fluorescence-activated cell sorting (FACS), the cells must first be detached from their substratum. However, enzymatic digestion or the usage of non-enzymatic cell dissociation buffers can result in the degradation of surface proteins, which might prevent their subsequent identification and cell separation in respective protocols. This makes it extremely challenging to use flow cytometry for phenotyping and characterization of adherent cells [13]. Trypsin, for example, is frequently used for detaching adherent cells. It time-dependently degrades most cell surface proteins by cleaving peptides after lysine or arginine residues that are not followed by proline [14]. This results in degradation of most surface proteins during cellular dissociation. Similarly, other enzymes such as extracellular matrix-specific collagenases, the serine protease elastase cleaving the peptide bond of C-terminal neutral, non-aromatic amino acid residues [15], the peptidase dispase hydrolyzing N-terminal peptide bonds of non-polar amino acid residues [16], and many other detachment agents provoke the significant break down of proteins.

Therefore, several milder enzyme mixtures such as Accutase and Accumax or non-enzymatic cell dissociation reagents such as a mixture of ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) chelating divalent metal cations have been introduced for routine cell passaging and manipulation of sensitive cells. These formulations are less toxic and preserve most epitopes for subsequent flow cytometry analysis [17,18].

More recently, the culturing of cells in a three-dimensional microenvironment has become the focus of researchers. These 3D cell cultures are either produced by culturing cells within a defined scaffold such as hydrogel or polymeric materials derived from extracellular matrix proteins or agarose or as self-assembly systems in which the cells grow in clusters or spheroids. It is well-accepted that these in vitro cell models offer the possibility to study cellular reactions in a closed system that better resembles the physiological situation than cell culture technologies that rely on two dimensions. As such, these models are particularly interesting for those studying aspects of cell-to-cell interactions, tumor formation, drug discovery, stem cell research, and metabolic interactions [19]. In comparison to 2D systems, 3D models have the potential to completely change the way drug efficacy testing, disease modeling, stem cell research, and tissue engineering research take place [19]. Finally, these systems will substantially decrease the use of laboratory animals in some research areas, which is a key aspect of the 3R principle [1].

3. Culture Media

Proper cell culture media are critical in the maintenance and growth of cell cultures and to allow the reproducibility of experimental results. Some cells additionally need non-essential amino acids (alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine) for effective growth and the reduction of the metabolic burden of cells.

The most common standard media used to preserve and maintain the growth of a broad spectrum of mammalian cell types are, for example, Dulbecco’s modified Eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) media. Typically, these media contain carbohydrates, amino acids, vitamins, salts, and a pH buffer system ( ).

Table 1

Inorganic salts/buffers: CaCl2: 0.2 g/L, Fe(NO3)3 × 9 H2O: 0.0001 g/L, MgSO4: 0.09767 g/L, KCl: 0.4 g/L, NaHCO3: 3.7 g/L, NaCl: 6.4 g/L, NaH2PO4: 0.109 g/LAmino acids: L-Arginine × HCl: 0.084 g/L, L-Glutamine: 0.584 g/L 1, Glycine: 0.03 g/L, L-Histidine × HCl × H2O: 0.042 g/L, L-Isoleucine: 0.105 g/L, L-Leucine: 0.105 g/L, L-Lysine × HCl: 1.46 g/L, L-Phenylalanine: 0.066 g/L, L-Serine: 0.042 g/L, L-Threonine: 0.095 g/L, L-Tryptophan: 0.016 g/L, L-Tyrosine × 2 Na × 2 H2O: 0.12037 g/L, L-Valine: 0.094 g/LVitamins: Choline chloride: 0.004 g/L, Folic acid: 0.004 g/L, myo-inositol: 0.0072 g/L, Niacinamide: 0.004 g/L, D-Pantothenic acid (hemicalcium): 0.004 g/L, Pyridoxal hydrochloride: 0.004 g/L, Riboflavin: 0.0004 g/L, Thiamine × HCl: 0.004 g/LOthers: D-Glucose: 4.5 g/L 2, Phenol red × Na: 0.0159 g/L 3, Pyruvic acid × Na: 0.11 g/LOpen in a separate window

Common media such as DMEM are available in a ready-to-use liquid form or alternatively in powdered media for easier storage and a longer shelf life. Moreover, many media can be obtained with different glucose concentrations (low or high glucose) as well as in formulations with and without L-glutamine or alternatively with stabilized glutamine. Finally, they are sold with or without a pH indicator such as phenol red.

Importantly, basal media typically contain no proteins, lipids, hormones, or growth factors. Therefore, these media require supplementation with fetal bovine serum (FBS), or often referred to as fetal calf serum (FCS), commonly at a concentration of 5–20% (v/v). FBS is obtained from the blood of fetuses of healthy, pre-partum bovine dams. The final serum is depleted of cells, fibrin, and clotting factors by centrifugation of the clotted blood. It should be noted that FBS from different sources might differ in growth factor and hormone profiles, virus content, endotoxin load, osmolality, total protein and metal content, sugars, and final processing (e.g., filtration, testing for potential contaminations). Therefore, most scientists prefer to buy traceable FBS batches with reliable lot-to-lot consistency to obtain reproducible results during experimentation.

In addition, newborn calf serum (NBCS) obtained from calves less than 20 days of age, calf bovine serum (CBS) sourced from calves aged between 3 weeks to 12 months, and adult bovine serum (ABS) isolated from adult cows more than 12 months old are frequently used to supplement cell culture media. Some researchers routinely heat-inactivate the serum at 56 °C for 15–30 min to inactivate the complement and to destroy potential bacterial contaminants. However, in most cases this is not necessary and should be omitted because heat inactivation also reduces the concentration or biological activity of growth factors that are required for proper cell growth.

However, it should be noted that serum-containing media has a number of disadvantages. Serum is complex, has an indefinite composition leading to batch-to-batch variation, increasing the risk of contamination, and the use of serum is commonly associated with ethical concerns in terms of avoiding the suffering of fetuses and animals [21,22]. Therefore, the development of serum-free media (SFM) has become a research hotspot during the last decades [21]. In principle, SFM can be divided into five types, namely (i) common SFM, (ii) xeno-free medium containing human-source but no animal components, (iii) animal-free medium, (iv) protein-free medium, and (v) chemically defined medium [21,23]. All these media contain key components (e.g., energy sources, vitamins, amino acids, lipids, trace elements, and inorganic salt ions) and are often enriched with special supplements such as anti-shear protectants, nucleic acids, and other ingredients that are required to improve the culture performance for certain cell types or applications [21]. Unfortunately, certain companies and suppliers of SFM often provide incomplete or no information at all about the composition of their media. Therefore, researchers already started a decade ago to install an online serum-free online database for the interactive exchange of information and experiences concerning SFM [22].

Biological contamination arising from bacteria, yeast, fungi, and mycoplasma can be better prevented by the addition of antibiotics and anti-mycotics to cell culture media. Most of them act by either inhibiting cell-wall synthesis (e.g., penicillin), interfering with membrane permeability (e.g., amphotericin B), or by inhibiting protein synthesis by preventing the assembly of the bacterial initiation complex between mRNA and the bacterial ribosome (e.g., streptomycin). However, the routine usage of antibiotics might develop slow growing persistent/resistant bacterial contaminants that may cause subtle alterations of cell differentiation and behavior [24]. In addition, antibiotics such as penicillin, streptomycin, and gentamycin can significantly alter gene expression and regulation and could modify the results of studies focused on drug response, cell regulation, and differentiation [25,26]. For example, a concise review has recently highlighted numerous publications that have shown the impact of antibiotics and antimycotics such as penicillin/streptomycin, gentamicin, and amphotericin B on in vitro properties of cells including proliferation, differentiation, survival, and genetic stability [27]. Similarly, a comprehensive literature search has found a number of reported side effects that are induced by different antibiotics, again supporting the notion that antibiotic-free culture media are recommended when possible to ensure the reliability and reproducibility of cell culture findings [28]. Consequently, researchers should avoid the permanent use of antibiotics in cell culture and should better try to implement strict aseptic working conditions to prevent bacterial contaminants in cell culture.

4. Phenol Red

Phenol red also known as phenolsulfonphthalein is the most frequent pH indicator in cell cultures. This water-soluble dye is a yellow zwitterion at low pH, while it changes to a red-colored anion or a fuchsia-colored di-anion at more basic conditions ( ). Therefore, this dye has been used as an inert pH indicator dye in many tissue culture media to detect pH shifts, waste products of dying cells, or overgrowth of contaminants that typically cause an acidification of the medium. However, based on its structural resemblance to some non-steroidal estrogens, it has the capacity to bind to estrogen receptors with an affinity of 0.001% of that of estradiol, thereby stimulating the proliferation of estrogen receptor positive cells [20]. Thus, it is advisable to dispense phenol red during experimentation when working with estrogen-responsive cell systems.

Open in a separate window

6. Short Tandem Repeat Profiling

Nowadays, the most common method to identify cross-contamination and cell misidentification is short tandem repeat (STR) profiling. This method can compare the number of allele repeats at specific loci in DNA between different samples. Although the respective allelic variants of these repeats are rather polymorphic, the number of alleles is very small. Therefore, multiple STR loci are analyzed simultaneously in a multiplex PCR assay for making different STR profiles effective for identification or discrimination purposes with a high discriminative statistical power.

In STR analysis, the amplified variable microsatellite regions obtained from the template DNA are separated on a genetic analyzer and subsequently analyzed with software that calculates the number of repeats at each variant site. Nowadays, effective and standardized STR panels are established for many species. In this regard, the Consortium for Mouse Cell Line Authentication that has established a multiplex PCR assay comprising 19 mouse STR markers is pioneering [66,67]. This multiplex PCR provides a unique STR profile for different mouse cell lines, including closely related cell lines. Representative chromatograms of four STR markers obtained for the widely used immortalized murine cell line AML12 are depicted in .

Open in a separate window

Importantly, when comparing the 19 mouse STR markers between three different mouse cell lines, each cell line has a unique constellation that allows the unequivocal discrimination of each cell line from the others ( ).

Table 5

STR MarkerGRXAML12N-HCC25 *STR 1-1 (MCA-1-1)101116STR 1-2 (MCA-1-2)161319STR 2-1 (MCA-2-1)9916STR 3-2 (MCA-3-2)141214STR 4-2 (MCA-4-2)19.320.320.3STR 5-5 (MCA-5-5)1514, 1517STR 6-4 (MCA-6-4)1915.318STR 6-7 (MCA-6-7)121217STR 7-1 (MCA-7-1)262926.2STR 8-1 (MCA-8-1)1614, 1516STR 9-2 (MCA-9-2)ND1518STR 11-2 (MCA-11-2)161816STR 12-1 (MCA-11-2)161917STR 13-1 (MCA-13-1)171517STR 15-3 (MCA-15-3)25.321.322.3STR 17-2 (MCA-17-2)1613, 1516STR 18-3 (MCA-18-3)162116STR 19-2 (MCA-19-2)121313STR X-1 (MCA-X-1)26, 272627Open in a separate window

Nowadays, databases are available in which primer information for the setup of STR testing for mouse, cat, dog, cattle, horse, men, and others are deposited [69]. Moreover, the Cellosaurus resource provides an incredible wealth of information and offers routines such as the STR Similarity Search Tool (Cell Line Authentication using STR, CLASTR) for comparing STR profiles [70].

7. Cell Line Alteration and Over-Passaging

Typically, immortalized cell lines are grown in the lab for many generations. However, a cell line cultured at high passage number or for prolonged times can show chromosomal duplications or rearrangements, mutations, and epigenetic changes [71]. This phenomenon is commonly known as genetic drift. Consequently, the morphology, proliferation rate, metabolic capacity, or general cell health can change dramatically, affecting experimental outcomes [72]. Therefore, the documentation of cell line passage number, which reflects the number of times the cells have been subcultured into a new vessel, is an important consideration when performing an experiment.

It was also reported that the passage number can increase the risk of viral contamination [73]. Furthermore, over-passaging of cells selects faster growing cells that in some cases show reduced secretion rates, carrier-mediated transport, and paracellular permeability, while having increased transcellular permeability [74]. Consequently, similar or even the same investigations performed in different laboratories might have completely different experimental outcomes when the passage number differs by hundreds of passages. Although there are no specific guidelines regarding the optimal passage range, common practice is to not use cells after 20 to 30 passages. Unfortunately, the precise knowledge of passage number is often not known, especially when the cells were obtained from a source other than a cell repository, which usually provides data on the cell passage number [75].

In some cases, researchers argue that even the passage number is imprecise because different laboratories may use different initial cell seeding densities or splitting rates during passing, which affect the number of times cells divide in cultures. Therefore, a formula for the calculation of precise population doubling level (PDL), which is synonymous with the cell generation time, was introduced, which is used particularly for primary cells. In the respective formula, the PDL, which is the total number of times the cells in the population have doubled since their primary isolation in vitro, is calculated as follows:

PDL = 3.32 (log Xe − log Xb) + S, where Xb is the number of seeded cells at the beginning of the incubation time, Xe is the cell number at the end of the incubation time, and S is the starting PDL before splitting [75,76,77].

8. Biosafety Aspects in Working with Cells

Cell cultures may have the ability to cause harm to human health and the environment and need to be assigned to a biosafety level that takes into consideration a multitude of factors [78]. Before working with a cell line, it is necessary to have an accurate knowledge about these risks, taking into account the intrinsic properties, type of (genetic) manipulation, and the resulting biological hazard inherent with the respective cell line that may be significantly increased by contaminating pathogens. Although these estimates must be conducted on a case-by-case basis, there are some general guidelines that need to be followed. First, the closer the genetic relationship of a cell under investigation is to humans, the higher the risk is to humans because contaminating pathogens usually have a specific species barrier. Nevertheless, care should be taken because some contaminating organisms have the potential to cross the usual species barrier [79,80,81]. Second, the tumor-inducing potential of a cell line is strongly dependent on its origin. While, for example, epithelial and fibroblastic cells have a low tumor-inducing potential, hematopoietic cells have a significantly higher one [82]. Third, well-characterized cell lines that are already used in many laboratories for many years have an overall lower risk than uncharacterized continuous growing cell lines or primary cells. After identifying and evaluating potential risks, it is essential to define ways of avoiding or minimizing these risks by containment, restricting the movement of staff and equipment into and out of cell culture laboratories, working according to standard operating procedures (SOPs), avoiding formation of aerosols or splashes during working, regular cell culture training, and by the implementation and following of the general guidelines of good laboratory practice (GLP) [2,78]. In addition, vaccination against Hepatitis B virus is advisable when working with primary human cell cultures. However, global norms and international standards for biosafety and biosecurity are often highly variable between different countries and should be noted before starting with the work [83].

9. Patient-Derived Cell Lines, Organoids, Xenograft Models, and Conditional Reprogramming

As discussed, established cell lines can undergo genetic drift or phenotypic alterations after long-term passaging. Therefore, they may no longer faithfully represent all the molecular features that were characteristic of the initial cell type they originated as. Consequently, scientists have developed techniques that allow the establishment of primary cells from either tissue or blood from healthy donors or subjects suffering from a defined disease. For humans, these patient-derived cell lines have high translational clinical relevance [84]. However, spontaneous immortalization is commonly a rare event and the establishment of respective cells was most often performed in the past by transformation with viral oncoproteins that partially deregulate the cell cycle or by overexpression of TERT that replaces short DNA segments that are lost during cell replication and are involved in control of cell senescence [6]. Similarly, the CRISPR-Cas9-mediated targeting of oncogenes that can be used to immortalize cells in vitro has been identified as an effective tool for establishing immortalized cell lines [7,8].

However, it should be critically noted that these manipulations can exert transcriptional and cell cycle effects and, further, that the inhibition of DNA damage signaling pathways by respective agents leads to the accumulation of mutations [85,86]. More recently, conditional reprogramming (CR) has emerged as a powerful tool for the establishment of long-term cultures of primary cells [86]. The technique of CR that was first established in 2012 allows the induction of normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro. In this technique, cells can be conditionally reprogrammed by co-culturing them with irradiated fibroblast feeder cells and the Rho kinase inhibitor Y-27632 [87]. This technology is now widely used to establish patient-derived cell cultures from both normal and diseased cells. In this procedure, the epithelial cells are reprogrammed to acquire an adult stem cell character by transferring the cells from standard culture medium to a CR medium that reverses their differentiation state and allows the generation of large numbers of cells for use in patient-derived models [87]. As such, CR offers exciting possibilities in precision medicine, regenerative medicine, drug testing, gene expression profiling, xenograft studies, and to define genetic, epigenetic, and metabolic alterations occurring during the transition from a normal to a tumor cell phenotype [88]. Importantly, sophisticated protocols are now available that further allow the use of CR for the rapid and efficient expansion of non-epithelial cells including those of neural, neuroendocrine, endocrine, and mesenchymal origin that conditionally can be grown for long periods [89].

In personalized medicine, organoids, which are self-organized 3D tissues typically derived from pluripotent fetal or adult stem cells, have gained enormous interest [90]. They are a kind of miniaturized and simplified version of an organ that forms in a selective 3D medium that includes a set of growth factors [91]. In particular, patient-derived organoids (PDOs) have been widely introduced in cancer research. They recapitulate basic features of primary tumors including histological complexity and genetic characteristics and are therefore ideally useful to predict the sensitivity toward antitumor drugs or aspects of tumor progression [92].

Similarly, patient-derived xenograft (PDX) models are dynamic entities in which cancer evolution can be experimentally monitored [93]. PDXs are cancer models established by implanting and growing a patient’s tumor cells in a suitable animal host. In most cases, the recipient is an immunodeficient mouse engrafted with a human immune system [94]. These models have become a useful experimental tool for the study of molecular interactions between human immunity and cancer cells. Particularly, these models have become highly attractive in basic research to understand aspects of cancer progression and metastasis. In addition, PDXs are frequently used in preclinical cancer research to identify novel predictive cancer biomarkers, test the efficacy of cancer drugs, investigate intra-tumoral heterogeneity and clonal dynamics, evaluate personalized therapy options, and to test the general translational hypothesis [94,95].

All these advanced cell culture techniques that more closely mimic the cellular microenvironment are nowadays an integral part of basic and clinical research. However, the usage of these patient-derived models (PDMs) requires extensive expertise, training, and quality control. To foster the development of respective models, several international consortia have been established with the aim to generate novel human tumor-derived culture models with associated genomic and clinical data. Representative initiatives have been launched by the National Cancer Institute (NCI) and the Human Cancer Models Initiative (HCMI) [96,97].

10. Cleaning and Sterilization

Biosafety depends on the cleanness of the laboratory. The general guideline is to strictly follow all possible safety rules. Ignoring or failing to follow any safety regulations can result in laboratory-associated infections and environmental contamination. Therefore, the biological risks need to be reduced by decontamination of biological agents that were used during laboratory operations.

In proper waste management, the inactivation methods should be appropriately validated whenever possible. In principal, there are four main categories for decontamination, namely (i) sterilization by heat, (ii) disinfection with liquids, (iii) disinfection by vapors and gases, and (iv) exposure to UV radiation.

Autoclaving is the most effective and reliable method to sterilize laboratory materials and decontaminate or inactivate biological agents. It is a sterilization method that uses high-pressure water steam and is the method of choice for decontamination of culture media, glassware, and pipette tips. However, it is essential that sufficient high temperature and pressure are maintained for a period of time that also guarantees spore inactivation. Typically, autoclaving for 60–90 min at 121 °C is sufficient to achieve a waste temperature of at least 115 °C for 20 min. Moreover, the operation and maintenance of autoclaves should be performed only by trained individuals and the success of autoclaving should be regularly checked by biological indicators. It is essential that waste or materials subjected to autoclaving are placed in containers or sealed autoclave bags that permit good heat penetration [98]. Hazardous chemical substances or radioactive waste should not be autoclaved. Contaminated scalpel blades, hypodermic needles, knives, and broken glass should be collected in puncture-proof containers with covers. Large volumes of liquid waste and contaminated media should be decontaminated before disposal in the sanitary water.

Chemical disinfection is usually the preferred method for decontamination of surfaces and furniture. For the optimal effectiveness of a disinfectant, several factors have to be considered. First, a disinfectant should have specificity for the biological agent to be removed. Second, a disinfectant should be suitable for the field of application because there can be significant activity differences when applied to surfaces or liquids. Finally, disinfectants may differ in their general application conditions (e.g., required contact time, working concentration) and in their effectiveness in the presence of other influencing factors (e.g., acids, organic load).

Similar vapors and gases applied in closed systems can provide excellent disinfection. Aerosols of hydrogen peroxide, chlorine dioxide, glutaraldehyde, paraformaldehyde, ethylene oxide, and peracetic acid are used in some laboratories to decontaminate biosafety cabinets or incubators [98,99]. However, all these chemicals are hazardous and disinfection with these substances should only be conducted by experienced and trained personnel.

Finally, exposure to ultraviolet (UV) radiation can effectively destroy most microorganisms. In particular, the UV-C light (spectral range: 100–280 nm), which is absorbed by the atmosphere, has the most destructive power for a wide spectrum of microorganisms [99]. Therefore, this UV region is often used in biological safety cabinets to reduce surface contamination [98]. However, the exposure to UV can cause burns to the eyes and skin of operators and should therefore be applied only with precaution. For cell culture beginners, it is therefore advisable to receive theoretical and hands-on training by experts before using UV-C radiation devices for cleaning and disinfection.

11. Conclusions

Cell culture plays an important role in biomedical research. However, cell misidentification, intra- and inter-species cross-contamination, and infection by bacteria, fungi, yeast, or viruses can lead to fatal consequences and pollution of the scientific literature. Therefore, regular testing for contamination and cell authentication testing should be mandatory in each laboratory working with cell cultures. Biological contamination with bacteria, molds, and yeast can be effectively removed by diverse bactericidal or fungicidal acting components. The ICLAC register of misidentified cell lines and the linked Cellosaurus knowledge resource are extremely important knowledge resources and provide helpful search tools such as CLASTR for comparing STR profiles. In addition, the implementation of good cell culture practice and aseptic techniques are essential to increase cell culture safety, promote the generation of reproducible scientific data, and facilitate comparability of results established in different laboratories. In addition, proper staff training and standardization of documentation and reporting of cell culture procedures are further effective means to promote high-quality work and safety in a cell culture laboratory. Recent advances in the generation of more physiologically relevant PDMs such as PDOs and PDXs have revolutionized common cell culture methods and helped to better understand human biology and pathophysiology. In this regard, CR is an attractive technique that can be used to rapidly and efficiently establish patient-derived cell cultures for basic and clinical studies and further significantly substitute animal-based research. Nevertheless, it should be kept in mind that the best designed and most well engineered cell culture laboratory is only as good as its least competent worker.

Funding Statement

RW is supported by grants from the German Research Foundation (grants WE2554/13-1, WE2554/15-1, and WE2554/17-1) and by a grant from the Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University (grant PTD 1-5).

Author Contributions

R.W. wrote the paper and S.W. arranged the final figures. S.K.S. provided B,C. E.M.B. provided electron microscopic images depicted in and . All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

More experimental details about original data depicted in , and will be made available by the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Want more information on chemistry flask type? Feel free to contact us.