Information

Origin of term 'confluency' in cell culture


Since as long as I have been doing cell culture, the word confluency has been used to describe the percentage growth of cells or area covered by them. However, no dictionary that I have found uses this word. I was wondering if anyone could reliably state where the meaning comes from or how the association began, and truly what it means. I don't believe I can post this on another This Site because the word doesn't have even a resembling meaning in the dictionary. Cell culture is such an integral part of cell biology, medical research and biological manufacturing. And this term is an integral part of cell culture. I was surprised to be unable to reliably verify its meaning.


Besides the etymologic explanation that @aandreev gave, in cell culture this term is commonly used to describe the density of adherent cells and it is used as a measure of their proliferation. It is usually combined with an estimated (or counted) percentage, so 10% confluency means that 10% of the surface the dish or flask used is covered with cells, 100% means that it is entirely covered. The cells grow twodimensional on the surface of the flask in this case. The picture below (from here) illustrates different levels of confluency:

The level of confluency is important as the cells change their growth with changing densities. Low density cells (10-20%) usually grow slower than 50% confluent cells. If the plate is complete grown by cells, they tend to grow much slower again. This influences their genetic program, behaviour in experiments and transfections.

As @Roland pointed out it is important to say that confluency is not a hard measure, but rather an estimate of the cell density. Different people will get different estimations, but the trend should be the same.


con- (com-) is prefix that usually means "togetherness", joining. Root fluency/fluent comes from latin fluere, to flow.

Source: Google's definitions for con- and fluency (information scraped from OUP).


The word confluency is a variant of the word confluence, although this variant only appears with any frequency in the context of cell culture [1]. The word confluence itself appeared in the fifteenth century and is derived from the Late Latin confluentia meaning “a flowing together” [2]. In English it was initially used for the flowing together of two or more streams, but acquired a large variety of more figurative meanings [3].

The word confluence (but rarely confluency) occurs in various biological contexts in the first half of the twentieth century [4], but the first example I can find in relation to monolayer cell culture is in 1961 [5], although the term confluent appears in the 1950s [6]. The form confluency seems to have appeared at about the same time as confluence [7]. Since the 1960s both forms have been used by different authors, currently with comparable frequency [8]. Some authors employ either one or the other exclusively, others use confluency when indicating a percentage designation and confluence to indicate the absolute concept - or vice versa [9].

The reason that confluency is not defined in general dictionaries (or even in the Oxford Dictionary of Biology [10]) can only be surmized. None of the online dictionaries I have consulted [11] give the cell culture usage as an example of confluence, suggesting that its use is less frequent than in other technical contexts, where the meaning is similar. With this usage of confluence not being thought to merit separate mention (irrespective of the importance or otherwise of cell culture) then the need to define an alternative form would not have arisen.

It should be added that there is there is a Wikipedia entry for confluency [12] but, despite its title, this uses the term confluence in its definition:

“In cell culture biology, confluence refers to the percentage of the surface of a culture dish that is covered by adherent cells. For example, 50 percent confluence means roughly half of the surface is covered, while 100 percent confluence means the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer.”

Notes

[1] The hits reported by a Google Book ngram for the word confluency between 1800 and 1954 were analysed individually, as journal results from ngrams often have erroneous dates. There was one example (1829) of its occurrence before 1900, and 13 before 1950, about half in relation to vaccines or biological phenomena.

[2] The etymology of confluence given in the online etymological dictionary is: early 15c., “a flowing together, especially of two or more streams,” from Late Latin confluentia, from Latin confluentem (nominative confluens), present participle of confluere “to flow together," from assimilated form of com “with, together” (see con-) + fluere “to flow” (see fluent). I would emphasize that the word confluence is not a compound formed in English by adding a prefix to the word fluency, but derives from the compound word, confluentia, already evolved in Late Latin (i.e. by the sixth century).

[3] The Oxford English Dictionary quotes an early instance of the extension of the meaning of the word confluence (1606) as “In this confluence of so many prosperous successes”. A variety of examples, appearing to come from the fields of politics, philosophy and mathematics, can be found in the Cambridge Dictionary online for its second definition of confluence, “a situation in which two things join or come together”.

[4] This can be seen by going to the websites of some of the older biological journals (e.g. Journal of the National Cancer Institute or the Journal of Experimental Medicine) and searching for the word confluence.

[5] 1961 Yearbook of the Carnegie Institute of Washington (p.66).

[6] For example W.R.Earle (of Earle's saline) uses it in a paper on the growth of fibroblasts in 1951, and T.T.Puck (of Puck's saline) uses it in a 1958 article in Experientia. The earlier use of confluent (also of 15th century origin) in relation to monolayer culture suggests that the word confluency, rather being a corruption of confluence, may have been formed from the adjective - perhaps in ignorance that a noun form already existed.

[7] Königsberg et al. (1960) J. Biochem. Biophys. Cytol. 8 333-343.

[8] There seems to have been division at the outset. In the US, Harry Eagle and Renato Dulbecco used the term confluency, whereas Howard Green and George Todaro used confluence. What one might refer to as the Glasgow school always used confluence: John Paul) at the Beatson Institute and his student Ian Freshney, both authors of influential books on cell culture. The more recent frequency of use may be judged from a Google ngram for “cell confluence v. cell confluency”.

[9] Searching for confluence in the Journal of Cell Science and examining recent issues brings up results for both confluence and confluency. This recent paper uses confluence exclusively both as an absolute concept and as a percentage, whereas this recent paper, for example, uses confluency exclusively in both absolute and numerical senses. Another paper refers to confluency, but to 70% confluence, but exactly the opposite is found in this paper which refers to confluence, but 70-80% confluency. Some papers - e.g. this alternate at random. The editors of the journal obviously do not care.

[10] The Oxford Dictionary of Biology has nothing between the entries for cone and confocal.

[11] Collins, Chambers, Cambridge, Lexico or Merriam-Webster.

[12] Wikipedia entry for confluency.


Tissue Culture: Definition, History and Importance

Tissue culture is the method of ‘in vitro’ culture of plant or animal cells, tissue or organ – on nutrient medium under aseptic conditions usually in a glass container. Tissue culture is sometimes referred to as ‘sterile culture’ or ‘in vitro’ culture. By this technique living cells can be maintained outside the body of the organism for a considerable period.

According to Street (󈨑) tissue culture is referred to any multicellular culture with protoplasmic continuity between cells and growing on a solid medium or atta­ched to a substratum and nourished by a liquid medium.

By plant tissue culture new plants may be raised in an artificial medium from very small parts of plants, such as, shoot tip, root tip, callus, seed, embryo, pollen grain, ovule or even a single cell, whether the cultured tissue develops into a plant or grows unorganized depends on the genetic potential of the tissue and the chemical and physical environment.

According to the parts used for culture the aseptic plant culture may be of follow­ing types:

(a) If a seedling is cultured it is called plant culture.

(b) When an embryo is cultured it is known as embryo culture.

(c) If plant organs, such as, shoot tips, root tips, leaf primordia, flower primordia or immature fruits are cultured, it is called organ culture.

(d) The culture of unorganized tissues from cell proliferations of segments of plant organs is called callus culture. Cell proliferations are formed in the explant due to injury caused by excision.

(e) When a single cell or small cell aggregate in a dispersed state is cultured, it is called cell suspension culture. It is also known as cell culture.

Culture of single cell is sometimes called single cell cloning. The portion of the plant to start the culture is called an explant. Culture derived from a single explant is called a clone. In order to maintain a culture for a comparatively longer period the culture me­dium is changed from time to time.

This process will remove those harmful excretory substances which have accumulated due to metabolism. By transferring a fragment of the parent culture to a new medium subculture is done. Such a fragment is called an inoculum.

History of Tissue Culture:

In 1832 Theodor Schwann said that cells could be cultured outside the body of the organism if provided with proper external conditions. In 1835 Wilhelm Roux cultured embryonic cells of chicken in salt solution. Reichinger (1839) said that fragments thicker than 1.5 mm were capable of growth but fragments below this limit failed to grow. He did not used any nutrient in his experiment.

Arnold (1885) and Jolly (1903) observed growth and cell division of leucocyte cells of salamander in culture. In 1907 American zoologist Ross Granville Harrison successfully cultured nerve cells of frog in solidified lymph. Harrison is known as the father of tissue culture.

M.J. Burrows (1910) cultured embryonic tissue of chicken in plasma. Mammalian cells were first cultured by Alexis Carrel. By repeated sub-culturing he was able to culture the tissue for 34 years. Organ culture was first done by D.H. Fell (1929) in England. He used solidified plasma and embryonic extract as nutrient medium.

History of Plant Tissue Culture:

German botanist Gottlieb Haberlandt first attempted to culture plant tissues ‘in vitro’. He started his work in 1898. He used cells from palisade tissues of leaves, cells from pith, epidermis and epidermal hairs of various plants for culture in media -containing Knop’s solution, aspergine, peptone and sucrose.

The cultured cells sur­vived for several months but the cells failed to proliferate.

This may be due to:

(a) Use of very simple media,

(b) Culture of highly differentiated cells and

(c) Aseptic techni­ques were not used.

In similar experiments by some later workers cells remained alive for a long period but they failed to divide.

Culture of meristematic tissue was started in early 20th century. Isolated root tips were first cultured by Robbin in 1922. Working independently Kotte (󈧚) also made similar observations. Robbin and Maneval (󈧛) cultured roots and maintained the culture for 20 weeks by sub culturing.

In 1934 White first successfully cultured isolated tomato roots in a medium conta­ining sucrose, inorganic iron salts, thiamine, glycine, pyridoxine and nicotinic acid etc. Gautheret (󈧦) noted that cambium culture from Salix capraea, Populus nigra etc. continued to grow for few months under aseptic conditions. He later (󈧩, 󈧪) used medium supplemented with B-vitamins and IAA.

In 1937 White recognised the importance of B-vitamins for growth of root cultures. Went and Thimann (󈧩) discovered the importance of auxin (IAA). Nobecourt (󈧩, 󈧪) obtained some growth in culture of carrot root explants. He also noted root differentiation in tissue culture. In 1938 tumour tissues of tabacco hybrid were succe­ssfully cultured.

In 1939 working independently three scientists, White in USA and Nobecourt and Gautheret in France cultured successfully plant callus tissue on synthetic medium continuously. Gautheret (󈧫) said that carrot culture required Knop’s solution supplemented with Bertholots’ salt mixture, glucose, gelatine, cysteine HC1 and IAA.

White (󈧫a) in culture of procambial tissue from young stem of the hybrid Nicotiana glauca × N. langsdorfii noted unlimited and undifferentiated growth. He showed that this tissue could be repeatedly subcultured. White (󈧫b) recorded development of leafy buds in tissue culture of the hybrid N. glauca × N.langsdorfii in nutrient medium.

Tissues from various plants were cultured subsequently. It was noted that older cultures show increasing degree of organization. The role of vitamins in plant growth was also recognized. Wetmore and Wardlaw (󈧷) successfully cultured shoot tips of pteridophytes (Selaginella, Equisetum and ferns).

Tissues of Sequoia semipervirens were cultured by Ball (󈧻). Pollens of Taxus and Ginkgo biloba were cultured by Tulecke (󈧿). Conifer tissues were successfully cultured by Harvey and Grasham (󈨉).

From tissue culture studies important information about root-shoot relationship can be obtained. Several scientists reported about the factors controlling vascular tissue differentiation from tissue culture studies.

Van Oberbeck (󈧭) cultured embryos of Datura on a medium supplemented with coconut milk. Importance of coconut milk and 2-4D as nutrient was recognised. The stimulatory pro­perty of coconut milk is due to the presence of zeatin.

The potent cell division factor was found to be kinetin, which is a 6 furfurylaminopurine. Cytokinin is 6-substituted amino-purine compound, which can stimulate cell division in culture of plant tissues. Monocot tissues were successfully cultured on a medium containing coconut milk.

Callus culture of Tagetus erecta and Nicotiana tabacum on liquid culture medium when agitated on a shaker produced suspension of single cells or cell aggregates (Muir 󈧹). Such cell suspension could be subcultured.

Studies on cell suspension cul­ture were carried out by Muir, Hildebrandt and Riker (󈧺), Street, Shigomura (󈧽), Torrey and Reinert (󈨁), Reinert and Markel (󈨂). Muir (󈧹) developed paper raft nurse technique for single cell culture.

In this method isolated single cells were put on a square filter paper, placed on a active nurse tissue, which supplies the required nutrients to the growing single cell. In another method cells were suspended on a han­ging drop in a micro-chamber.

Bergman (󈨀) working with suspension cultures of Nicotiana tabacum var. sansum and Phaseolus vulgaris var. ‘early golden rod’ developed agar plating technique of single cell cloning. In this me­thod single cell fraction was separated by filtration, mixed with warm agar and then plated in a petridish in thin layer.

Melchers and Bergmann (󈧿) noted that after several cultures of the haploid shoot of Antirrhinum majus there was increase in ploidy. Ball (󈧲) noted the possibility of regeneration of whole plant in culture of shoot tip of angiospermic plants. Wetmore and Wardlaw (󈧷), Morel (󈨀) obtained whole plants from culture of shoot apices having 1 or 2 leaf primordia. Morel (󈨄) used this method for culture of orchids.

A cell which can develop into a whole organism by regeneration is called a to­tipotent cell. This term was coined by Morgan in 1901. According to White (󈧺) if all the cells of a multicellular organism is totipotent, then such cells in isolated condi­tion regain their dividing power and can produce whole plants. In an organism this capacity remain suppressed.

It was noted that single cells are capable of producing new plants. From pollen and anther culture haploid embryos were obtained. A method of microspore culture of Nicotiana and Datura was developed by Nitsch (󈨎, 󈨑). He was able to double the chromosome number and obtained homozygous diploid plants.

From anther culture of tobacco Bourgin and Nitsch (󈨇), Nakata and Tanaka (󈨈) obtained haploid tissues and haploid embryoids.

Cocking (󈨀) recorded release of protoplasts from root tip cells by using fungal cellulase in 0.6M sucrose. He was able to culture isolated protoplasts, which regenerate new cell walls and produce cell colonies and ultimately plantlets.

In many plant suspension cultures cell protoplasts had been successfully released. Plant tissue culture technique is used for the study of tumour physiology.

White and Brown (󈧮) were able to culture bacteria free crown gall tumour. In Scorzonera hispanica Gautheret (󈧲) noted that the callus culture which initially required auxin, produced some proliferations which can grow in auxin deficient medium.

Such inheri­ted changes occurring in the nutritional requirements (especially involving auxin) of cells of a culture is called habituation. An auxin habituated culture does not re­quire the supply of exogenous auxin (Butcher 󈨑). Butcher noted (󈨑) that when auxin and cytokinin habituated tissues are grafted into a healthy plant, tumours are produced.

Pathogen free plants can be obtained by culturing apical’ meristem.

By late 70’s it was evident that plant tissue culture technique can be successfully used in various field of agriculture, such as, production of pathogen free culture, pro­duction of secondary products, clonal propagation, mutant culture, haploid breeding and genetic engineering.

By tissue culture, pathogen free cultures have been produced. This technique is im­portant for plant pathological investigations. Protoplasts in culture are used for virus infection and biochemical studies.

From suspension culture secondary products can be synthesised in large amount. Some of these substances are enzymes, vitamins, food flavours, sweeteners, anti-tu­mour alkaloids and insecticides. In Japan ‘in vitro’ culture has been achieved at industrial level.

Clonal propagation of orchids and several other ornamental and economic plants have been achieved by ‘in vitro’ culture. In potato clonal propagation has been achieved by culturing leaf cell protoplasts. By using mutagens in culture followed by selection disease resistant or stress resistant mutant plants have been regenerated.

By haploid breeding few cultivers were produced. Hybrids of related but sexually incompatible species have been produced by protoplast fusion. By this technique hybrid between potato and tomato has been produced. By cell fusion of isolated cells from two different species hybrid tobacco plants are produced.

Dormancy period of seeds can be shortened by excising the seeds and culturing its embryo on artificial medium (embryo culture). Abortive embryo can be grown successfully by embryo culture.

Foreign genes with desirable characters attached to a plasmid may be inserted into the naked protoplast usually by means of liposomes. The expression of introduced gene in the mature plant is still doubtful.

Importance of Tissue Culture:

Tissue culture has great importance in studies of plant morphogenesis, physio­logy, biochemistry, pathology, embryology, cytology etc. From tissue culture studies it is possible to know bow simple cells differentiate and become specialized to perform special functions. Various changes taking place in a cell can be noted from clonal culture.

Interrelationship between two cells can be studied in tissue culture. With the help of phase contrast cine-photomicrography a very clear understanding of mitosis and meiosis is possible in tissue culture. Haberlandt noted the importance of tissue culture in studying plant morphogenesis. Relationship between growth and differentiation can be well understood from such a culture.

In vegetatively propagating plants many plantlets are formed very quickly from callus culture or culture of explants. Orchids, which normally propagate very slowly, can form many plantlets very rapidly in shoot tip culture. This is also noted in car­nation.

By tissue culture method new plant variants can be obtained by isolating gene­tically unique cells. From callus cultures of tobacco, carrot, asparagus etc. new plants are formed. Such plants show genetic variability.

From studies of mutant cells, the biochemical and developmental process of an organism can be better understood. In tissue culture, mutation can be easily induced and from such mutant cells mutant plants may be produced.

In tobacco, paddy etc. from anther culture haploid plantlets are produced. By doubling the chromosome homozygous plants are obtained most rapidly. So, this process has immense importance in plant breeding.

Tissue culture technique has been successfully used in nutritional research. The effects of various mineral salts, vitamins etc. on growth may be studied in culture. Many important information about glucose metabolism, nitrogen metabolism and hormone production can be obtained from ‘in vitro’ culture.

Suspension culture under controlled conditions may be used to solve many physiological or biochemical problems and also provides a system for the production of important plant products, such as, plant alkaloids.

From cell and organ culture under controlled environmental conditions nutri­tional and metabolic processes can be studied. Some mutant cells cannot grow in a medium which does not contain a special nutrient. From this biochemical steps of a process can be determined.

Tissue culture has great significance in pathological studies. The effect of various medicine on cells infected by pathogens can be studied in tissue culture.

Culture of maize cells from plants susceptible to the race T of Helminthosporium maydis were treated with pathotoxin of the fungus. Scientists were able to obtain cells resistant to this fungus. From such cells resistant plants were also produced.

Tissue culture technique is employed in the studies of plant tumour diseases and host parasite relationship. Disease free plants can be produced by tissue culture te­chnique. Tissue culture has great importance in vaccine production. In 1949 vaccine for poliomyelitis has been produced after observing that the poliomyelitis virus can attack human cells. Later vaccines for mumps, meseales, and influenza have been pro­duced.

The process of virus attack, effect of virus on Post cells and how new viruses are produced etc. have been studied in tissue culture. The behaviour of substances, which can prevent virus attack has been studied on virus infected cells.

In tissue culture the behaviour of normal and cancer cells can be studied. It has been noted that some viruses and carcinogenic chemicals can produce cancer. Effect of radiation and chemicals on normal and cancer cells has been studied. From such studies it may be possible to know which chemical substances can destroy cancer cells.

From tissue culture studies information about some hereditary diseases of man has been obtained. Carriers of some diseases can also be identified through tissue cul­ture technique. From leucocyte culture the cause of mongolism in man has been dis­covered. From such culture abnormal Philadelphia chromosome has been identified. This chromosome has some relation with chronic granulocytic leukomia.

When tissue transplant is done from one person to the other then sometimes there is tissue rejection. So it is necessary to match the tissue of donor and receiver before actual transplant. This can be done by culturing the mixed leucocytes of the donor and the receiver.

Distantly related species usually do not hybridize. This difficulty can be omitted by cell fusion and protoplast fusion technique. Carlson in 1972 successfully produced hybrid plants by protoplast fusion between Nicotiana glauca X N. langsdorfii. Power (󈨐) obtained hybrids between Petunia hybrida and P. parodii by protoplast fusion.

Kaw and Wetter (󈨑) produced hybrids between tobacco and soyabean by cell fusion. Thus cell fusion and protoplast fusion techni­ques have great importance in plant breeding. Tetraploid fertile Lolium and Festuca hybrids were obtained by somatic cell fusion.

Those embryos which fail to produce mature fruits normally can be cultured and from such embryo cultures plants are produced. Embryo culture also prevents seed dormancy. Cooper (󈨒) obtained hybrid plants between barley and rye by embryo culture.

Conservation of Germplasm:

By tissue culture plant germplasm can be stored.

This method can be success­fully used to solve various problems:

(a) Many seeds, such as, seeds of Citrus sp., Coffea sp., Hevea brasiliensis etc. retain their viability for a short period. These can be conserved by tissue culture.

(b) Vegetatively propagated plants (such as, banana, potato, sweet potato, and yam) which do not produce seeds or which are highly heterozygous, are stored as cuttings or tubers. This requires much labour charge and are expensive to propa­gate. This problem can be solved by tissue culture.

Many fruit trees of Rosaceae are propagated by budding, grafting and layer­ing. By tissue culture rapid propagation of such plants are possible.

(c) In many economic plants, such as, coconut, date plam etc. vegetative pro­pagation normally does not occur. The germplasm of such plants can be conserved by tissue culture.

(d) Many trees reproduce very slowly. By tissue culture such paints can be multiplied rapidly and many plants with parental genotypes are formed.

For conservation of germplasm the cells should be stored in such a condition which allows minimum cell division. One of the method attempted is storing of cells in liquid nitrogen having a temperature of — 196°C.

For germplasm conservation shoot tips or plantlets can be stored. Such stored materials can be used as and when required.

In tissue culture cell division can be suppressed by various methods:

(i) To the medium growth retardant may be added. The substances used are absicic acid, mannitol, sorbitol, malic hydrazide, succinic acid etc. Potato shoots are successfully stored in a medium containing malic hydrizide.

(ii) Low temperature is helpful for storage of cells in culture. Cultures of potato, sweet potato, beet, grape, apple, etc. can be stored by this method. Temperate crops (e. g. potato) are stored usually at a temperature of 0—6°C and tropical crops (e.g. sweet potato) at 15—20°C. By this method meristem culture of strawberry has been conserved for six years.

(iii) The concentration of nutrients of the culture medium may be changed. Some substances required for normal growth may be supplied at a lower concentra­tion or may not be supplied at all.

(iv) The gas composition within the culture vessel may be changed. The atmos­pheric pressure or oxygen concentration may be lowered to conserve the cells.

Production of Secondary Metabolic Products:

Some plants produce secondary metabolic products, such as, alkaloid, anti­biotic, glycoside, resin, tannin, saponin, volatile oil, etc., which are of considerable economic importance.

By cell culture various secondary metabolites (e.g. allergin) have been synthesised artificially. Cultivation of plants producing secondary metabolites can be improved significantly by tissue culture. There are certain disadvantages in the production of secondary metabolites by tissue culture.

(i) In cell culture synthesis of secondary metaboli­tes occur at a lower rate than in an entire plant,

(ii) After prolonged culture ‘in vitro’ the production of secondary metabolites may decrease or even stop,

(iii) The cost of large scale production of secondary metabolites in cell culture is high. So, only very rare and expensive secondary metabolites are produced by tissue culture.


When to subculture?

The criteria for determining the need for subculture are similar in adherent and suspension cultures however, there are some differences between mammalian and insect cell lines.

Cell LinesCell densityExhaustion of medium
Mammalian cellsAdherent cultures should be passaged when they are in the log phase, before they reach confluence. Normal cells stop growing when they reach confluence (contact inhibition), and it takes them longer to recover when reseeded. Transformed cells can continue proliferating even after they reach confluence, but they usually deteriorate after about two doublings. Similarly, cells in suspension should be passaged when they are in log-phase growth before they reach confluency. When they reach confluency, cells in suspension clump together and the medium appears turbid when the culture flask is swirled.A drop in the pH of the growth medium usually indicates a buildup of lactic acid, which is a by-product of cellular metabolism. Lactic acid can be toxic to the cells, and the decreased pH can be sub-optimal for cell growth. The rate of change of pH is generally dependent on the cell concentration in that cultures at a high cell concentration exhaust medium faster than cells lower concentrations. You should subculture your cells if you observe a rapid drop in pH (>0.1 – 0.2 pH units) with an increase in cell concentration.
Insect cellsInsect cells should be subcultured when they are in the log phase, before they reach confluency. While tightly adherent insect cells can be passaged at confluency, which allows for easier detachment from the culture vessel, insect cells that are repeatedly passaged at densities past confluency display decreased doubling times, decreased viabilities, and a decreased ability to attach. On the other hand, passaging insect cells in adherent culture before they reach confluency requires more mechanical force to dislodge them from the monolayer. When repeatedly subcultured before confluency, these cells also display decreased doubling times and display decreased doubling times, decreased viabilities, and are considered unhealthy.Insect cells are cultured in growth media that are usually more acidic that those used for mammalian cells. For example, TNM-FH and Grace’s medium used for culturing Sf9 cells has a pH of 6.2. Unlike mammalian cell cultures, the pH rises gradually as the insect cells grow, but usually does not exceed pH 6.4. However, as with mammalian cells, the pH of the growth medium will start falling when insect cells reach higher densities.

You will get useful cell numbers in various sizes of tissue cell culture dishes, plates and flasks. We provides useful numbers, such as, growth surface area, volumes of dissociation EDTA-Trypsin solution, culture medium, seeding density and cell numbers at 100% confluent, are given below for Greiner Bio One and Nest Biotechnology cell culture dishes, cell culture plates and cell culture flasks. This can be a reference for cell culture dishes, plates, flasks from other companies, such as Corning, TPP, ThermoFisher Scientific, Sigma, etc according to the same cell growth surface area.

In mammalian tissue cell culture, confluence is commonly used to estimate the number of adherent cells in a culture dish, plate or a flask, referring to the proportion of the surface which is covered by cells. For example, 70% confluence means roughly 70 percent of the growth surface is covered by cells, and there is still room for cells to grow. 100% confluence means the cell growth surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer.

Different cell lines exhibit differences in growth rate. Most cells are typically passaged before becoming fully confluent in order to maintain their proliferative phenotype. Some cell lines are not limited by contact inhibition, such as immortalized cells, may continue to divide and form layers on top of the parent cells. To achieve optimal and consistent results, experiments must be conducted at certain confluence, depending on the cell type. The cell number listed in the growth chart here is based on Hela cells, and provided as a reference. For your specific cell type you may have to gain empirical numbers.


Terminology

Compiled by the 1990 Terminology Committee
Members: Dr. Stephen Mueller,
Dr. Michael Renfroe, Dr. Jerry W. Shay, and Dr. James Vaughn
(Originally published in In vitro Cell. Dev. Biol. 26:97-101, January 1990)

When areas of research become interdisciplinary their jargon, techniques and bodies of information are utilized widely in diverse disciplines. At such times, reciprocal use of terminology by individuals who had not previously used it is often misused and, therefore, confusion occurs. This confusion is especially acute in the field of vertebrate, invertebrate and plant cell culture. There is hardly a field of biological investigation in which culturing of such cells is not employed. Similarly, molecular biology and molecular genetics are lending their technology to an ever widening group of researchers who are communicating in a more global sense via scientific presentations, publications and research proposals. Unfortunately often the writer and reader represent different areas of specialization who have been brought together by the common technology used in their work. As such, misuse of terminology can prove unfortunate indeed anything from inability to repeat a piece of research to problems in publishing a paper or obtaining funding of a research proposal. The following glossary, approved by the Society for In Vitro Biology, Terminology Committee is published in an effort to increase communication between scientists and between scientists and the lay community.


Abstract

Mechanical phenotyping of adherent cells has become a serious tool in cell biology to understand how cells respond to their environment and eventually to identify disease patterns such as the malignancy of cancer cells. In the steady state, homeostasis is of pivotal importance, and cells strive to maintain their internal stresses even in challenging environments and in response to external chemical and mechanical stimuli. However, a major problem exists in determining mechanical properties because many techniques, such as atomic force microscopy, that assess these properties of adherent cells locally can only address a limited number of cells and provide elastic moduli that vary substantially from cell to cell. The origin of this spread in stiffness values is largely unknown and might limit the significance of measurements. Possible reasons for the disparity are variations in cell shape and size, as well as biological reasons such as the cell cycle or polarization state of the cell. Here, we show that stiffness of adherent epithelial cells rises with increasing projected apical cell area in a nonlinear fashion. This size stiffening not only occurs as a consequence of varying cell-seeding densities, it can also be observed within a small area of a particular cell culture. Experiments with single adherent cells attached to defined areas via microcontact printing show that size stiffening is limited to cells of a confluent monolayer. This leads to the conclusion that cells possibly regulate their size distribution through cortical stress, which is enhanced in larger cells and reduced in smaller cells.


Cell culture basics

Culture conditions


For cell survival and proliferation, it is essential that the culture environment replicates, as best as possible, the physiological environment of cells. Culture conditions that can be controlled include temperature, relative humidity and CO2 levels as well as factors associated with the media, such as nutrient composition, pH, osmolality and the volume and frequency of replenishment. These variables can fluctuate over time so they should be monitored. Table 3 highlights the optimal culture conditions for most mammalian cell cultures, however exceptions do exist. 5

Table 3: Optimal cell culture conditions for most mammalian cells. 1 , 2 , 4 , 8


6. Large-scale retrieval of stem cells for clinical applications

(a) Cell detachment

Cell detachment is a critical step for subculturing surface adherent cells. At a small scale, mechanical detachment methods are well established for different types of cells and are traditionally used for PSCs. However, for large-scale cell expansion these methods cannot be used. In principle, the efficiency of cell detachment depends on the cells to be detached, the microcarrier and carrier surface, and thus the interaction of the cells with the microcarrier and, of course, also the detachment agent. Traditionally, proteolytic enzymes, in particular, trypsine or its recombinant and/or animal-free substitutes, are used for the efficient generation of single cell suspensions.

In addition to genomic instability seen to be readily generated in systems where enzymatic dissociation is used [138], there are further disadvantages when using proteases for cell detachment. Goh et al. [90] showed that trypsin was the most efficient detachment agent with respect to speed and efficiency for detaching human fetal MSCs from Cytodex 3 carriers, followed by collagenase I and TrypLE Express with an overall viability of the detached cells of more than 95% for all agents. However, all of the cells detached with protease showed (osteogenic) differentiation efficiency significantly lower than that observed for the non-treated/non-harvested cells the differentiation efficiencies were 26� µg Ca ++ -deposition/10 6 cells (i.e. indicator for osteogenic differentiation) and 71 ± 4 µg Ca ++ -deposition/10 6 cells, respectively.

This indicates very clearly the negative impact of protease treatment for cell detachment on cell fate due to the degradation of ECM proteins, destruction of cytoskeletal structures and cell-to-cell contacts. Similar effects caused by proteolytic cell detachment, for instance, have been reported by Canavan et al. [139] for the detachment of bovine aortic endothelial cell monolayers. Specifically for hMSCs, Potapova et al. [140] reported a decrease of CD105 expression with time of exposure to trypsin over a range of 5� min. These results clearly indicate that the exposure to trypsin or any other proteolytic agent should be as short as possible. Based on this observation, Nienow et al. [141] evaluated a novel two-step protocol for trypsinization of hMSCs comprised of the addition of and incubation with trypsin-ETDA for 7 min at a fivefold increased agitation rate (30 → 150 r.p.m., followed by 200 r.p.m. for the last 5 s) leading to an increase in the maximal specific energy dissipation rate (ɛ) from approximately 9 × 10 𢄤 to 0.11 W kg 𢄡 . This equates to a reduction of the Kolmogorov length from about 180 µm (well above the eddy size at which damaging occurs, as suggested by Croughan et al. [31,36]) to 55 µm, which is well above the size critical for isolated cells, however sufficient to detach cells. The second step consisted of the inactivation of trypsin and the removal of the carriers via filtration. Nienow et al. [141] reported a recovery of more than 95% of the cells which maintained their specific characteristics.

With respect to the detachment of hiPSCs, there is only limited literature available. In principle, when aiming to get to a single cell suspension, trypsin or equivalent (recombinant) proteases, including Accutase, Accumax or TrypLE, are preferable because the protein bridges between the cells are also degraded. However, the generation of single cell suspension of hiPSCs is characterized by high cell loss due to dissociation-induced apoptosis after passaging as single cells, which could be solved by the use of Rho kinase inhibitors, such as Y-27632 or ROCKi [142]. Another way to circumvent this problem is dissociation into aggregates because it has been shown that microcarrier cultures of PSCs can also be seeded with cell aggregates/clumps and that single cell suspensions are not required [143]. The only issue of importance is the fact that the size of aggregates has to be uniform during the culture, which is achieved by agitation generating the required turbulences (see above).

Dissociation of cultures into aggregates can be achieved by using other proteases and agents, including collagenase IV, dispase or hypertonic Na-citrate solution. In this context, Nie et al. [106] showed that the use of hypertonic Na-citrate solution led to superior results with respect to viability and total viable cell number obtained over several passages when compared with detachment using collagenase, dispase or mechanical methods (order of decreasing efficiency). The cells detached using the hypertonic citrate solution kept their ability to express pluripotency markers and could differentiate to all three germ layers after 30 passages.

Thus, in general, the use of non-proteolytic cell detachment means (absence of proteases) has an undeniable positive effect on subcultivation and differentiation of stem cells. These few references show very clearly that any proteolytic cell detachment should be avoided in order to maintain the organization of the cellular cytoskeleton as well as the cell-to-cell interaction as much as possible and to reduce cell damage. For regenerative medicine purposes, protocols for non-proteolytic cell sheet detachments have been developed (e.g. [144]), mainly based on the use of a temperature-responsive poly(N-isopropylacrylamide (pNIPAAm) modified polystyrene surface allowing the detachment of cells by a reduction of temperature (below the lower critical solution temperature) leading to hydrophilic surface properties and cell detachment [145]. Since, no proteases are used, the cells detach as cell sheets because the intercellular proteinous bridges are preserved. In order to improve cell adhesion and proliferation such thermo-responsive surfaces can be functionalized, including grafting on heparine for binding bFGF [146], the cell-binding domain RGDS [147] or the cyclic RGD peptide CRGDC [148] because of its high affinity binding to the αv㬣, αv㬥 or 㬕㬡 integrins of cells, to mention just a few possibilities.

Only recently, microcarriers grafted with poly(N-isopropylacrylamide allowing the non-invasive harvesting of anchorage-dependent cells had been evaluated. That this principle can be implemented was shown by Kenda-Ropson et al. [149] and Tamura et al. [150] for the detachment of CHO-K1 from modified microhex carriers (pNIPAAm-grafted 2D-polystyrene carriers) and from chloro-methylated polystyrene beads (pNIPAAm-grafted), respectively. Furthermore, Yang et al. [151] and Gümü𕿞relioğlu et al. [152] showed the efficient detachment of human bone marrow-derived MSCs from Cytodex 3 grafted with pNIPAAm and of mouse fibroblasts (L929) and human keratinocytes (HS2) from cross-linked poly(2-hydroxyethyl methacrylate) (PHEMA) beads grafted with pNIPAAm. In all cases, the reduction of the temperature from 37ଌ to less than 30ଌ led to cell detachment, though more in the form of cell clumps than isolated cells. Although this is a very promising development, few advances have been performed in this specific domain with respect to thermo-responsive microcarriers and further efforts are necessary in order to solve many still existing technological hurdles before large-scale implementation.

With respect to the use of such temperature-responsive culture supports for the expansion of iPSCs, it is highly probable that they can be used for this type of cells without any drawbacks, since their differentiation potential as well as genetic stability is not affected by temperature reduction and exposure to 25ଌ and 4ଌ for up to 2 days [153]. More details on the possibilities and use of thermo-responsive and, in general, of stimuli-responsive cell culture supports can be found in the review by Brun-Graeppi et al. [154].

(b) Separation of non-differentiated cells and of carriers: problem of carrier breakage

Removal of undifferentiated cells is a particular concern in clinical applications of differentiated progenies derived from iPSCs because undifferentiated PSCs are teratogenic [78]. On a small scale, fluorescence activated cell sorting for the isolation of differentiated cells, such as cardiomyocytes [155], neural [156] or endothelial cells [157], can be used. A similar technique is immunomagnetic cell sorting [158], allowing separation at a larger scale. The selective removal of undifferentiated cells using cytotoxic antibodies, such as the cytotoxic mAb 84 [159], is also of high interest and can be coupled to immunomagnetic cell sorting [158] reducing further the risk of teratoma formation. In certain cases, such as the separation of PSC-derived cardiomyocytes from non-cardiomyocytes and undifferentiated cells, the separation is enabled by distinct metabolic flow. Tohyama et al. [160] reported that in a glucose-depleted and lactate-enriched medium only cardiomyocytes survived and that no tumours were formed after transplantation. More extended information can be found in recent reviews by Chen et al. [15], Abbasalizadeh & Bahavand [161] and Diogo et al. [162]. Nevertheless, it has to be mentioned that to date no systematic studies on harvesting PSCs and separation their differentiated progenies have been performed.

Another important issue should be touched here very briefly: Gupta et al. [163] found that 𠆎xcessive’ stirring of microcarrier (Cytodex 3) cultures (r.p.m. > 25) in Bellco spinner flasks can lead to bead breakage. This is a known problem for soft carriers like Cytodex 1 or 3, but is also of concern when the expanded and differentiated cells are used in vivo, because these preparations have to be free of any residual microcarrier or sub-particles derived from these carriers. Sieving is a principle way to get rid of entire carriers however, breakage products are removed with difficulty or not at all. There are two main potential solutions to this problem: either using rigid carriers which are resistant to breakage, like beads made from polystyrene or glass, or using soft carriers which can be dissolved using proteases, such as gelatin- or collagen-based carriers. However, it should be recalled that the choice is critical because the rigidity/stiffness of the support may have an influence on the stem cell fate (see above). On the other hand, it is evident that aggregate cultures without using carriers as an initial support would also present a solution for this specific problem.


Cell confluence in cell cultures - myoblast c2c12 in particular (Feb/01/2011 )

Hi to all. I'm a grad student and I work on cell cultures. I'd like to ask you one thing about the tecnique, I hope you can help me to understand sorry if my english is not perfect.

I work with the myoblasts cell line c2c12. I know that cells must not reach confluence becouse otherwise they begin differentiate. furthermore they must be at 60-80% confluence before passing or splitting them.

Does the term "confluence" mean that cells cover the entire surface of the plate?
How can I see when cells reach 60-80% confluence? Do you know if I can find somewhere some images about cell coltures at different grade of confluence?

Thank you very much for your precious help!

Correct, confluency is the amount of culture surface that is covered by cells. Your best bet for learning how to assess the confluency is to get an experienced person to look at your cells and tell you how confluent they are and learn from that.

You can think of it like this. 25% confluent is a quarter of the surface area covered by cells, 50% is half the area and 75% is 3/4 of the area.


Types of Animal Cell Culture

On the basis of cell growth and division, the animal cell culture is subdivided into the following two types:

Primary Cell Culture

It is defined as the cell culturing from the tissue of the host animal. The cells can be obtained directly by the mechanical method and indirectly by the enzymatic action. Once, the cells are obtained, they have to be cultured on a suitable container that must be provided with all the nutrients required for the cell division and growth. The growth of primary cells either occurs as an adherent monolayer on the solid media or as a suspension in a liquid medium.

  1. Adherent cells: These cells adhere to the solid surface and produce a cell population in the monolayer pattern. Adherent cells are sometimes called a confluent cell, in which the cells merge or contract to fill the surface area. Fibroblasts and epithelial cells are examples of adherent cells. The properties of the adherent cells include:
    • Adherent cells are anchorage-dependent.
    • Grows in a monolayer pattern.
    • Growth occurs on the solid surface or we can say on the solid media.
    • Adherent cells fill the entire surface area of the container or vessel as a monolayer.
    • Adherent cells follow the property of contract inhibition, in which the cell itself ceases the growth of some cells to maintain the synchronized state through chemical signalling. Once a monolayer is formed, the formation of new cells is inhibited by the adherent cells.
  2. Suspension cells: These are the cells that do not adhere to the surface of the medium. Suspension cells are the type of cells that float as the suspension over the liquid medium. The properties of the suspension cells include:
    • Suspension cells are anchorage-independent.
    • Floats as a suspension of cells over the liquid culture medium.
    • Growth occurs in the liquid nutrient medium.
    • Suspension cells grow much faster than the adherent cells.
    • There is a short lag phase in the suspension cells.
    • Enzyme action is not required for the dissociation of cells.

Secondary Cell Culture

It is defined as the subculturing of the primary cells, which later produce secondary cells lines. The passaging or subculturing of the primary cells result in a phenotypic and genotypic uniformity of the cell population. After the subculturing, the cell-lines will become different from the original cells. Based on the cell’s life span, the cell lines can be categorized into:

  1. Finite cell lines: Here, the cells possess a limited life span and show a limited cell division. Passaging value is less because the cells after some time lose the ability to grow or proliferate and enter into the phase of senescence or ageing.
    Example: Normal cells produce finite cell lines.
  2. Continuous cell lines: In continuous cell lines, the number of cell division and passaging value is indefinite. The passaging value is more because the continuous cells do not lose the ability to divide, i.e. these can grow and divide by an infinite number of times.
    Example: Cancerous cells produce infinite or continuous cell lines.

Process

The process of animal cell culture can be summarized into the following series:

  1. Tissue explant: It involves the removal of tissue from the organ.
  2. Cell extraction: It is carried out either mechanically or enzymatically. The extraction is mostly carried out by the enzyme action or by the process of trypsinization.
  3. Culturing in a nutrient medium: After that, the cells are cultured either on a solid nutrient medium or liquid nutrient medium. The primary cells form a monolayer over a solid nutrient medium, whereas appears as a cell suspension over the liquid medium.
  4. Subculturing: It is also called cell passaging. The subculturing is carried out after the formation of primary cells and it is important to continuously study or to grow the cells. This is the most important step in cell culture, which helps us to understand the cell type.

Normal cells: These cells possess a low passaging value because they lose their ability to divide after some time due to cell ageing. Therefore, the cell divides to produce definite cell lines.

Continuous cells: These cells possess a high passaging value because the cells have the ability to continuously divide. Therefore, the cell divides to produce indefinite cell lines.

Passaging value is directly proportional to the cell type and cell division.

  • For continuous cells, the passaging value increases, by which the cell division will be more.
  • For normal cells, the passaging value decreases as the cell ages, by which the cell division capacity will also decrease.

Difference Between Normal and Continuous Cells

  • The normal cell produces a monolayer, whereas the continuous cell does not.
  • The continuous cell does not follow the property of contract inhibition, while the normal cells follow.
  • A normal cell has a property to divide for definite times, whereas the continuous cell has a property to divide again and again.
  • The continuous cells possess a high passage value, whereas the normal cells possess a low passage value.
  • The capacity of dividing in the continuous cell remains the same as the first passaging, whereas the capacity of dividing decreases as a result of cell ageing in the normal cells.

Advantages

  • Animal cell culture makes the use of a low amount of reagents.
  • Serial passaging maintains the homogeneity of the cell types.
  • Provides controlled physiological conditions.
  • Provides controlled physiochemical conditions like temperature, oxygen concentration, ph etc.

Disadvantages

  • Animal cell culture requires high technical skills to interpret and to regulate the animal cell culturing.
  • It is a very expensive method to carry out.

Applications

The animal cell culture provides a model to study the effects of drugs, biochemistry etc. of the cell. By animal cell culture, we can perform tissue engineering. It also helps us to identify the cancerous cell as both the normal and continuous cells can be grown in the animal cell culture.

We can also study the replication and life cycle of the virus, so it plays an important role in the field of virology. In animal cell culture, we can also study the effect of different drugs on different cell types, so it also helps in toxicity testing.


2 - Cell culture techniques

Cell culture is a technique that involves the isolation and maintenance in vitro of cells isolated from tissues or whole organs derived from animals, microbes or plants. In general, animal cells have more complex nutritional requirements and usually need more stringent conditions for growth and maintenance. By comparison, microbes and plants require less rigorous conditions and grow effectively with the minimum of needs. Regardless of the source of material used, practical cell culture is governed by the same general principles, requiring a sterile pure culture of cells, the need to adopt appropriate aseptic techniques and the utilisation of suitable conditions for optimal viable growth of cells.

Once established, cells in culture can be exploited in many different ways. For instance, they are ideal for studying intracellular processes including protein synthesis, signal transduction mechanisms and drug metabolism. They have also been widely used to understand the mechanisms of drug actions, cell–cell interaction and genetics. Additionally, cell culture technology has been adopted in medicine, where genetic abnormalities can be determined by chromosomal analysis of cells derived, for example, from expectant mothers. Similarly, viral infections can be assayed both qualitatively and quantitatively on isolated cells in culture. In industry, cultured cells are used routinely to test both the pharmacological and toxicological effects of pharmaceutical compounds. This technology thus provides a valuable tool to scientists, offering a user-friendly system that is relatively cheap to run and the exploitation of which avoids the legal, moral and ethical questions generally associated with animal experimentation.


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