**4.2 pH value**

pH plays a vital role in cell culture. The cell growth rate is decline associated with Fluctuations in pH level. That's why routine monitoring is essential. For the cells is 7 the optimal pH, and decline or increase in pH can hinder the growth of cells. More decrease in pH level (usually in between 6.0–6.5), can stop the growth rate of the cells, and cells are start losing viability at low pH level. That's why pH level should be maintained and monitored carefully for individual cell line. If pH level fall rate is less than 0.1 units/day, that indicates the cell condition is good and no need to hurry to change the culture medium immediately. If it is 0.4 units/day (pH drop rate), that indicates the culture medium need to be changed quickly [13]. Alkali (like- NaOH, KOH) or acid (HCl) solution helps to control pH level in culture medium. Besides, NaHCO3 (sodium bicarbonate) or natural buffer solution, and addition (need base) of CO2 gas to the bioreactor also helps to maintain optimum pH level in culture medium. Generally, pH electrode (silver chloride electrochemical-type) used within the bioreactor [14]. For the proliferation of cell in culture medium an optimum, stable as well as balanced pH is essential. Depending upon cell type and culturing process pH level may vary and specific. Generally, 5–10% CO2 required to grown cells using buffered media that contain NaHCO3 (sodium bicarbonate) and where maintained the range of pH 7.2 to 7.4 [15]. CO2 incubator, optimum pH level, ideal temperature, optimum moisture condition, sterile and clean working environment are essential to maintain and complete an experiment. In the cell culture medium, the carbonate buffer helps to hold constant pH and take parts in releasing CO2 gas in the CO2 incubator. Color of the culture medium depending and changing with the pH level of culture medium. Color indicates altering the medium and CO2 levels [3]. Commonly, 4–10% of CO2 is practiced in the cell culture technique. By maintaining HCO3 − concentration and CO2 tension in culture medium one can easily achieved optimum pH and osmolality [16]. By observing the color of the media can easily identify the pH condition like- Phenol red to yellow/ orange color indicates too acidic where pH 6.8 (bellow), Red to pink color indicates pH above 7.0 to 7.7 which is normal, and bright fuchsia color indicates pH 8.0 to 8.2 (too alkaline).

#### **4.3 Effect of temperature**

Generally, cell culture need 37°C for incubation called control temperature. Proliferation and multiplication of cells are significantly decreased at more than 40°C, like 41°C or 42°C temperature, and increased temperature may also cause high apoptotic rate of CEF cells. Cell viability, apoptosis, proliferation, and oxidative status of cells in culture medium can be altered with high temperature. ROS (reactive oxygen species) formation increased with increasing Temperature (Proportionally) [17]. During the transportation must be care full about temperature. Besides regular or routine inspection is recommended for better results. In cell culture technique one of the most challenging issues is to grow cell, that's why an ideal temperature play a vital role in cell culture along with good supplementation of nutrients. For the cell division the optimum temperature should be vary on cell type that assist to maintain growth rate. Generally, at optimal temperature metabolic function of cell is optimum as well as good that helps to increase their size, and proliferation rate [18]. Temperature requirement varies based on cell type like- *S. frugiperda* (sf-21, *Trichophusia ni* (Tn-5), *S. frugiperda* (sf-9) (insect) need 27°C, Chinese Hamster Ovary (CHO) (Mammalian)-need 36–37°C, Human embryonic kidney (HEK- 293 T) (Human) need 37°C, HF 205 and HF 210 (plant) need 27°C.

#### **4.4 Incubation**

A device in which microbial culture or cell culture is grown and maintained with customized temperature, humidity (relative humidity 95%), oxygen, CO2 level, and other conditions. For virus cultivation, cell culture as well as cell infection, vaccine development incubation is a very much crucial and fundamental issue. The incubator is an essential instrument in cell biology research, Microbiology, Biotechnology, Molecular biology research. On the other hand, an egg incubator is one of the most important for embryonated egg production in the laboratory and large scale. BOD (Biological Oxygen Demand) incubator is popular in this regard. There are so many necessary points always bear in mind to maintain good incubation. Firstly, cleaning inside, cabinets, outside and handle that helpful to eradicate cross-contamination, also helps to hold a good quality of cell, media, SPF (Specific Pathogen Free) eggs, and other chemicals or ingredients. Logbook maintenance is another vital issue to maintain high-quality research work. Through logbooks, laboratory personnel can easily identify any issue related to the incubator. A dedicated power supply essential to maintain cell quality, growth, metabolism as well as important to maintain cell physiology. CO2 level and the supply are very much essential to maintain moisture and pH (normal range is 7.2 to 7.4). Generally, 5% CO2 with 37°C is used to maintain the cell. Eventually, inventory management will helpful for GMP (Good Manufacturing Practice) and proper cell culture as well as laboratory work.

#### **4.5 Cryopreservation**

Cryopreservation is the method in which intact living cells are conserved as intact in liquid nitrogen at cryogenic temperatures. On the other hand, in cryopreservation system using low temperature helps to protect living cells structurally intact. Freezing system keeps the living cells frequently for a long time (often for years). Freezing temperature ceased their typical metabolic activity that's why cells are protected from damage caused by long time preservation, and chemical reactivity. Gently handle

#### *A Brief Concept of Cell Culture: Challenges, Prospects and Applications DOI: http://dx.doi.org/10.5772/intechopen.99387*

cells because cell may be damaged and will get stress during the freeze–thaw process. Optimal cryopreservation of cells relies on proper freezing and thawing methods. A successful cryopreservation method calculating based on recovery rate of cells (frozen) from low temperature, percentage of alive cells, and rate of cells that function as normal after thawed [19]. Basically, for cryopreservation harvest the cells in exponential growth. Then gentle centrifugation done at 125 × g for 10 minutes. After that check and scree the media. The procedure starts with taking freezing medium (GM-which warm at room temperature for 30 minutes) that containing a cryoprotectant such as Dimethyl sulphoxide (DMSO) (e.g. 5–10% v/v DMSO), fetal bovine serum (FBS) (10–20% v/v) and at high cell density (1–5 × 106 /ml) and sometimes added knock-out serum replacer (KoSR; 20% v/v final), bovine serum albumin (5% final) or human serum albumin. From some recent research [20–22] it is said that freezing rate has great impact on viability of cryopreserved cells. It is suggested that cells be slowly cooled (like 2°C, −20°C, −80°C for at least 24 hours and finally preserved in liquid nitrogen at −196°C) that gives better surviving rate of cell in the cryopreservation process. Record logs must maintain during all the steps. From the final cryovial (that contain cell at −196°C) remove one vial and restore the cells in culture medium to determine cell viability and sterility. Recovery rate of cryopreserved cells depends on the types of cultured cell. Some cell needs several days, some shows low viability on the day of culture, in some cases cell produce debris, some cells are shows normal viability after 24 hours' post-thaw. Before retrieving of cryopreserved cells clean the biosafety cabinet, prepare the CCF, media, FBS and arrange all the instruments and appliances (sterile). Then wash the cryovial with 70% ethanol and place it in a water bath for 2 to 5 min at 37°C to melt. Transfer the thawed cell in a tube and gentle centrifugation (10 minutes at 125 × g) needs to discard (supernatant) cryoprotectant in the meanwhile collect the cell pellet and suspend the cells in 1 mL or 2 mL of complete growth medium (GM) then proceed for cell count and subculture in new CCF for 24 hours' observation. In the process of cryopreservation, significant rate of cell survival and maintenance of cell integrity (structural and morphological) can be achieved by using cryoprotective agents (CPAs). Excipient is an ingredient added intentionally to the drug substance which should not have pharmacological properties in the quantity used. Commercially available CPAs namely Dimethyl sulphoxide (DMSO) is commonly used as CPA. Factors behind the success of cell survival [23] are: (a) Type and concentration of cryoprotectants (an additive, such as glycerol or dimethyl sulphoxide, that can protect cells against freezing injury). (b) Cell density in cryopreservation solution at the time of freezing. (c) Cooling and thawing rates of cell suspension. (d) Dilution rate of thawed cell suspension. The main advantages [24] of cryopreservation are easily found original cell lines from the safety stocks, preserve the cells for year after year and lastly smoothly perform continuous research or experiments.

### **4.6 Thawing**

Correctly thawing of cells is crucial to recover quickly, yielding the highest viability and functionality possible. Some cryoprotectants (e.g. DMSO) has toxic effect on cells, due to the possibility of toxicity cells should be thawed rapidly and not allowed to remain in the freezing medium no longer than required time. Firstly, retrieve the cryovial containing the frozen cells from liquid nitrogen (−80°C or − 196°C freezer) and immediately place it into a 37°C water bath (for 1 to 2 min) or place immediately in a pre-equilibrated thermo-conductive rack or tube module resting on dry ice to

minimize cell warming/thawing. Rapidly thaw the cells (< 1 minute) by carefully swirling the cryovial in the 37°C water bath up to there is a little bit of ice left within the cryovial. Then place the cryovial into a BSC. Gently wipe the outside of the cryovial with 70% IPA (Isopropyl Alcohol) prior to open the cryovial screw. Carefully add (dropwise) required amount of pre-warmed GM into the tube (centrifuge) that containing recently thawed cells. Place the cell suspension in centrifuge machine for centrifugation and set 200 × g for 5–10 minutes (it may vary based on cell type). Inspect the transparency of supernatant and visibility of a pellet at the bottom after completing the centrifugation. Discard the supernatant aseptically without breaking the pellet. Softly resuspend the cells by gently pipetting with GM and prepare required concentration, then transfer the cell suspension into the CCF, and place it in the suggested culture environment. Inspect the cells using an inverted microscope for morphology. Examine an aliquot of cells for the ability to exclude trypan blue. If cells pass both inspections, they are ready for culture [25].

#### **4.7 Passaging of cells**

Subculture of cell commonly known as passaging of cells and the ratio of subculture is 1:2. The main concept of passaging: cells are split into half in each subculture. Continuous cell lines should will be passaged with higher split ratio due to their higher replication rate. Usually the number of times the cells have been subcultured into a new CCF known as passage number. In the case of diploid cell cultures, the number cell passage is partially equal to the number of population doubling level (PDL) since the culture was begun. On the other hand, PDL of continuous cell lines is not fixed like diploid cell culture. Mostly the PDL is an estimation or prediction. PDL may ups and down with cell stress and cell death (due to necrosis, apoptosis). Loss of proliferation capacity of cell, contamination of culture medium may also responsible. A common formula for the calculation of population doubling level: PDL = 3.32 (log Xe – log Xb) + S; where Xb is the cell number at the beginning of the incubation time, Xe is the cell number at the end of the incubation time, S is the starting PDL. Another common formula used to calculate the population doubling level is [26]: Log (*N/N*) x 3.33**;** Where N is the number of cells in the culture vessel at the end of a certain time interval, and N is the original number of cells plated in the vessel, Population doubling and passage number are often mixed up or thought to mean the same thing. The passage number describes the number of times that a culture has been subcultured The population doubling time is important to know when the number will be double and passaging will be required. According to ATCC [24] the population doubling time calculate with the formula: DT = T ln2/ln(Xe/Xb); where T is the incubation time in any units, Xb is the cell number at the beginning of the incubation time, Xe is the cell number at the end of the incubation time.

#### **4.8 Trypsinization**

Trypsin is an enzyme that is used to remove adherence proteins from a cell surface. Generally, trypsin-based disaggregation so-called trypsinization. Disaggregation of cells from the CCF commonly crude trypsin used, the effect of raw (crude) trypsin can easily be neutralized by commercially available serum (FBS) or trypsin inhibitor. On the other hand, pure trypsin is also used in the cell degradation process which is less toxic and very specific in action [27]. Commonly

## *A Brief Concept of Cell Culture: Challenges, Prospects and Applications DOI: http://dx.doi.org/10.5772/intechopen.99387*

0.05% trypsin used in laboratory work. Sometimes trypsinization causes cell damage and sometimes may not effective for some cells thus other dissociation agents (enzyme) are recommended for the dissociation of cells. Warm and cold trypsinization are the two common approaches. An extensively used method is warm trypsinization. In which cells are washed with basal salt solution and then add warm trypsin (37°C) adequately and stirred properly. The supernatant dissociated, the cells are dispersing in the medium. In the case of cold trypsinization, cellular damage is reduced, resulting in a high yield of viable cells also improved survival rate. For cold trypsinization cells are maintained in ice after washing with media or salt then treated with cold trypsin for 6–24 h. After that remove and discard the trypsin and incubate the CCF at 37°C (for 20–30 min). Dispersion of cells may start and fully dispersed cells counted using hemocytometer then dispersing in a medium for further use. The easiest way of trypsinization is a). Discard the media from CCF b). Wash the cell surface with PBS (4 ml for 75 cm<sup>2</sup> CCF) c). Take trypsin (Room temperature) d). Rinse monolayer of cell with trypsin–EDTA (2 to 4 ml for 75 cm<sup>2</sup> CCF) e). Stay for 2 minutes then discard trypsin and Incubate the CCF at 37°C for 5 minutes f). Tapping the CCF and collect the cells by scrapper stored in a tube g). Spin down the cells, resuspended by adding the growth medium or fresh medium, and Count the cells h). Split into a new flask and Incubate at 37°C.

#### **4.9 CCF measurement**

CCF denotes a Cell Culture Flask. It is also known as a tissue culture flask. CCF is important for culturing of cells, transportation of cells, and media. There are a lot of different volumes of CCF used for research work. Commonly, used flask volume are 25 cm<sup>2</sup> , 75 cm<sup>2</sup> , 175 cm<sup>2</sup> , 225 cm<sup>2</sup> , 300 cm<sup>2</sup> . Commonly 75 cm<sup>2</sup> CCF preferable for laboratory work. Cell contamination generally appeared during cell culture laboratory. Proper knowledge of CCF handling can minimize crosscontamination, which improves the quality and physiology of the cell. Rough handling of CCF during media transfer, passaging, scrapping will be responsible for the different vital issues. Mycoplasma contamination is one of them. Prompt and Improper pipetting during cell harvest and split from CCF may cause stressful conditions on cells and resulting in cell death. Scrapping of the cell for subculture or infection or cell count gently handles the CCF, corkscrew. Mild flame spark on corkscrew (CCF) by flame gun or gas burner helpful to safe the cell and flask environment. Covering the cork with Paraflim very much essential to save the cell. IPA (Isopropyl Alcohol) spray must be done before and after handling of CCF. After application of IPA, then CCF, media, FBS (Fetal Bovine Serum), tips, flame gun, trypsin other materials, and appliances allow entering into BCS (Biosafety Cabinet) for further processing. Some points should be bear in mind regarding CCF such as, is CCF allow pipettes, tissue scrapper, tissue spatula properly? Is CCF has marked on both sides? Is there any leakage? Is there any crake on cork? Is the bag of CCF tightly pack? Is the CCF clean (inside)? These points might be helpful for cell culture.

#### **4.10 Cell suspension**

The cell suspension is nothing but suspension culture. It's another type of cell culture where a small amount/volume of cells is permitted to grow in growth media forming suspension called cell suspension. If the cells are derived from other cultures or homogenized tissue, then use suspension culture. Both suspension culture and adherent culture are the same.

#### **4.11 Cell count and cell infection**

Cell counting was performed using a hemocytometer (Neubauer improved counting chamber, Precicolor HBG, Germany) or MacMaster slide and Trypan blue exclusion every 24 h (1,1 mixture of 0.2% Trypan blue in normal saline solution and sample). After placing the stock cell suspension on the hemocytometer and place a coverslip on it. Count cell of 4 (16 × 4) site, then the average of 4 sites of hemocytometer and count the total cell as = Average (number of the cell) × 10,000 × 2. Cell culture flask such as 25 cm<sup>2</sup> contain 5 to 10 ml culture media, 75 cm<sup>2</sup> contain 10 to 30 ml culture media, and 175 cm<sup>2</sup> contain 40 to 150 ml culture media. One (1) cm<sup>2</sup> need 90,000 cells likewise 75 cm<sup>2</sup> need 75 × 90000/5 = 13,50,000 cells minimum. Viable cells are considered as unstained ones and while stained cells are considered as dead under the inverted microscope. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell based assays. Cell counts must be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Cell infection is required for virus propagation, bulk antigen production. MOI rate is very much essential in cell infection. There are three types of MOI commonly used in the laboratory such as 1 MOI, 0.1 MOI, 0.001 MOI where 1 MOI means one (1) virus can infect one (1) cell. 0.1 MOI denotes 10 cells infected by one (1) virus and one virus can infect 100 cells in 0.001 MOI. Generally, practice 0.1 MOI means one virus is enough to infect 10 cells.

## **4.12 PFU (PFU), ELD50, EID50, MOI, CCID50, TCID50**

Plaque forming units (pfu) is an assessing of the total number of infectious virus particles. It is ascertained by a plaque-forming assay. In the field of virology study, a plaque-forming unit (PFU) is a measurement of the number of particles capable of forming plaques per unit volume i.e. virus particles. It is a functional measurement rather than a measurement of the absolute quantity of particles: viral particles that are defective or which fail to infect their target cell will not produce a plaque and thus will not be counted. For instance, a solution of virus with a concentration of 1,000 PFU/μl indicates that 1 μl of the solution contains enough virus particles to produce 1000 infectious plaques in a monolayer cell, but no inference can be made about the relationship of pfu to the number of virus particles.

ELD50- Embryo Lethal Dosage. ELD50 unit is the amount of virus that will kill 50 percent of inoculated eggs.

EID50- Embryo Infective Dosage. EID50 unit is the amount of virus that will infect 50 percent of inoculated eggs.

Multiplicity of infection (MOI) is the average number of virus particles infecting each cell. MOI is related to pfu by the following formula: Multiplicity of infection (moi) = Plaque forming units (pfu) of virus used for infection/number of cells. For example, if 2x106 cells is infected by 50 ml of the virus with a titer of 108 pfu/ml. The moi will be 0.05\*108 /2\*106 = 2.5. The fraction of cells that are not infected is P(0) = 1 - e−moi. To ensure 99% of cells are infected requires moi > 4.6. Assume the conditions used for plaque assay and TCID assay do not alter the expression of infectious virus.

TCID50/ml and pfu/ml are related by pfu/ml = 0.7 \* TCID50. As a working estimate, one can use pfu/ml = 0.5 \* TCID50 [28].

CCID50: Cell culture infectious dose which will infect 50% of the cell.

TCID50 is the tissue culture infectious dose that will infect 50% if the cell monolayers are challenged with the defined inoculum. Two methods commonly used to calculate TCID50 (can also be used to calculate other types of 50% endpoint such EC50, IC50, and LD50) are a). Spearman-Karber [29] b). Reed-Muench method.
