Overview of Primary Cell Culture Models in Preclinical Research of Prostate and Bladder Cancer

Abstract

The number of patients diagnosed with prostate and bladder cancer is increasing worldwide and one of the most important challenges remains the development of effective, safe and economically viable antitumor drugs. Clinical approval for drugs tested in preclinical studies enabling them to enter phase I clinical trials is essential. Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. Under the right conditions and with appropriate controls, authenticated cancer cell lines retain most of the genetic properties of the cancer of origin. Studies conducted during the initial development of drugs such as toxicity, corrosion and drug activity were carried out on animals; however, in the past two decades, alternatives have been sought due to the fact that animals do not effectively model to human in vivo conditions and unexpected responses are observed in the studies. Also, more than 100 million animals were used and billion dollars were spent for animal toxicity experiments. Cell culture studies made positive contributions to the initial development of drugs and is highly desirable, as it provides systems for ready, direct access and evaluation of tissues. Contrary to animal studies, less cost and the need for low drug and a short response time are the characteristics for in vitro cell culture methods. In vitro tumor models are a necessary tool, in not only the search for new substances showing antitumor activity but additionally for assessing their effectiveness. This chapter reviews the main features of primary cancer cell cultures, provides an overview of the different methods for their selection and management, and summarizes the wide range of studies that can be performed with them to improve the understanding of prostate and bladder cancer preclinical treatment processes.

1. Introduction

Cancer is the one of the major death cause worldwide and accounted nearly 10 million deaths in 2020 [1]. The rate of incidence in prostate cancer and bladder cancer are increasing worldwide too. According to GLOBOCON 2018, prostate cancer is the second most frequent cancer and in men, it is fifth leading cause of death. Bladder cancer is also common in men ranking on sixth position and ninth leading cause of cancer death [2]. There are so many treatments available like radiotherapy, chemotherapy, hormonal therapy but these treatments are associated with adverse side effects and poor quality of post treatment life. Hence there is need in development of effective, safe and economically viable antitumor drugs.

Prostate cancer and bladder cancer are heterogeneous diseases where many molecular, environmental and genetic factors are involved in its progression and understanding the mechanism of this progression is difficult [3]. In recent years the cancer research has made significant progress, but many challenges remain as it is [4]. Currently, only 7% of potential anticancer drugs are gaining approval which is much lower than drugs for other diseases [5]. Hence, to improve this percentage, it is essential to clinically approve drugs which are tested in preclinical studies and enabling them to enter phase I clinical trials [6].

Experimental models are important tools in the cancer research. The model should be reproducible, able to successfully reflect disease stage that is being studied and mimic the disease; how it behaves in humans [4]. Cell lines are in vitro model systems, a necessary tool, in not only the search for new substances showing antitumor activity but additionally for assessing their effectiveness. They are widely used in different fields of medical research and pharmaceutical companies. Presently pharmaceutical industries mostly rely on in vitro models like two dimensional (2D), three dimensional (3D), boyden’s chamber (to study chemotaxis and assessment of cell motility) [7], micro fluidic systems (It is small devices that can provide a specific fluid flow, constant temperature, fresh medium, flow pressure and chemical gradients which is same as in vivo systems to study migration and invasion [8], 3D bioprinting (mimics the processes that occurs in tumor micro environment) [9, 10]. Main reason for accepting in vitro model is it’s physiological relevance, it helps in improving the understanding of prognosis and treatment, it provides accuracy and it is also a low cost screening tool for researchers [11]. The usefulness of in vitro models is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. The in vivo model involves animals which provide valuable information to understand many aspects in development of disease and initial development of drugs such as toxicity, corrosion and drug activity [12]. But from past two decades, alternatives have been sought due to the fact that animals do not effectively model humans in in vivo conditions, as it shows unexpected responses like anatomical variation and also difficulty in extracting quantitative mechanistic data in the studies. Mathematical models are also used in the cancer research to analyze tumor growth and progression, and helps in predicting the effects of some therapies [13]. Different clinical setting, cancer resistance and switching to another treatment, existence of unknown biological details these issues can affect the mathematical models [14, 15, 16]. Computer simulation is another model in the cancer research, helps to test complex multi scale cancer progresses, it also accounts for drug pharmacokinetics and pharmacodynamics, but has drawback in less common cancers because of less data, therefore it lacks perspective validation and accuracy [17].

All models involved in the cancer research have pros and cons hence the cost duration, experimental design and data analysis in developing the anticancer drug should be considered for the selection of the model. It is necessary to choose more effective preclinical platforms to screen the antitumor compounds [18]. Practically in-vitro models of tumors will not only give primary screening of potential antitumor drugs but it also prevents drugs with insufficient antitumor activity from entering into preclinical animal testing [19]. This chapter reviews the main features of primary cancer cell cultures, provides an overview of the different methods for their selection and management, and summarizes the wide range of studies that can be performed with them to improve the understanding of prostate and bladder cancer preclinical treatment processes.

2. Primary cell culture

Primary cell culture is a gold standard testing platform for in-vitro research in oncology as they reflect the tumor state more accurately compare to most commonly employed cell lines [20]. It is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment [21]. Cell culture studies have made positive contributions to the initialdevelopment of drugs. Contrary to animal studies in vitro method requires low drug dose and short response time, which is characteristic feature of in vitro cell culture methods [22]. Primary cell culture is also called as ex-vivo. Because primary cells are directly taken from tissue origin and cultured under favorable conditions hence it is more similar to the in-vivo state and exhibit normal physiology. It maintains the cross talk between malignant and healthy components [23]. This is the main reason why primary cell culture is called as excellent model system to carry studies in metabolic, aging, signaling studies and effects of drugs, toxic compounds in the cell.

Primary cells are non-immortalized and non-transformable as they imitate the appearance of living model and hence these can help to model 3D tissues. In this culture, cells will grow in 3D aggregates and presents interesting application [24]. It helps for detection, isolation, growth and developmental stages of viruses and helps to analyze the mode of infection. Drug candidate and its toxicity screening rely on results of early-stage in vitro cell based assays. Particularly in pharmaceutical industry primary cell culture is used to synthesize verity of biomolecules in high scale, various research project on cell-based product are developed. It is alternative for animal model to test effect of drugs and cosmetics [25]. There are few technical hurdles associate with primary cell culture. For instance, culturing might be difficult if the quality of the surgical material is poor. Also, due to early onset of cell senescence, difficulty arises to maintain sufficient number of cell passages but researchers have made many attempts to resolve this problem [26, 27].

Primary cell culture has been subdivided in to adherent cell culture and suspension cell culture. In the adherent cell culture, cells are arranged in monolayer and attach to the surface of the culture flask. Adherent cells are usually derived from tissues of organs. Growth is limited to surface area and it needs to be detached from the surface before cells get sub-cultured. Viral vaccine, gene therapy and cell therapies depend only on adherent cell culture. Suspension cells are derived from the peripheral blood and do not require any attachment for growth. They do not get attached the surface area of the cultural flask. The cells are free floating and growth is limited to the concentration of cells. The steps involved in the primary cell culture are represented in (Figure 1).

Figure 1.

Flowchart showing the process of primary cell culture technique.

3. Isolation of cells

Before going to any further tissue processes, it is important to keep in mind that all tissue processing has to be carried out in a biosafety cabinet and all the sterilization protocols has to be maintained properly [28]. Now moving towards cell isolation, it is a process where one or more specific cells are isolated from heterogenous cell mixture. Isolation of primary cells from cancer cells is an important phenomenon of cell culture biology as they are more reliable sources to understand the human cell. There are many standard protocols available for culturing the normal andneoplastic cells [28]. Human prostate and bladder are composed of many cell types which can be isolated and cultured. Hierarchy of the epithelium has been reviewed most [29, 30]. There are 3 main epithelial lineages namely neuroendocrine, basal and luminal. Prostatic homeostasis is mainly depends upon the epithelial cells and stromal cells; stromal cells guides to the epithelium cell for their dedifferentiation, proliferation and also progression of carcinogenesis [31].

Now, how to understand which cell is cancerous and which cell is non- cancerous because cell does not contain tags on it. There are specific cell markers (Antigen) which will identify the difference between cancerous and non-cancerous for ex. ARA70 (Androgen receptor-associated protein 70) is a cell marker which was noted to be expressed at high levels in normal primary cultures compared with prostate cancer cell lines [32]. Many cell markers are available depending upon cell type which is listed in Table 1. Also in bladder cancer depending upon cell type there are different CD (Cluster of Differentiation) cell markers which are depicted in Table 2 [38]. Identification of stem cell marker has uncovered a cellular hierarchy of epithelium during development and in response to injury [39]. Cell markers (Antigens) have specific antibodies and these have to be evaluated histochemically. These reactions are evaluated by specific kits which are available in market (Table 3). For isolation, first tissues will be collected from prostate cancer patients and bladder cancer patients who are undergoing biopsy. This collection of tissues needs to be well coordinated between urology, pathology and the investigator and it has to proceed for primary culture within 2 hours after collection of tissues [33, 34, 35, 36, 37, 39, 40, 41]. After collection, tissue should be placed in sterile container which has HBSS (Hanks’ balanced salt solution) with HEPES (Hydroxyethyl piperazineethanesulfonic acid) and store at 4°C for 2 hours to increase cell viability. To asses tumor cells in the dissected material Hematoxylin and Eosin (H&E) stain is used in the histopathology lab. To get a single cell suspension from tissue dissociation obtained after sugary, there are three mechanisms available for isolation: Chemical, Mechanical and Enzymatic method (Table 4).

Cell marker	Cell type
Cytokeratin 5 [33]	Basal cell
CD59 [34]	Basal cell
c-Met [35]	Basal cell
CD95 [34]	Basal cell
Cytokeratin 8 [36]	luminal cell
CD9 [37]	luminal cell
15-LOX-2 [36]	luminal cell
CD24 [34]	luminal cell

Table 1.

Biomarkers for Prostate cell culture.

Cell marker	Cell type
CD9 [38]	Urothelial
CD104+ [38]	Basal
CD13+ [38]	Stromal cells of the lamina propria
CK5 [37]	Basal
p63 [37]	Basal
CK8 [37]	Luminal

Table 2.

Biomarkers for bladder cell culture.

Antigen	Antibodies	Cell type
Cytokeratin 8/18 (Prostate)	Mouse IgG1	Luminal cells and intermediate cells
High molecular weight cytokeratin (Prostate)	Mouse IgG1	Intermediate cells
Trop2-APC (Prostate)	Mouse IgG2a	Epithelial cells
CD49f-PE(Prostate)	Rat IgG2a	Epithelial cells
CD + 9 (Bladder)	antibody clone M-L13	Urothelial
CD104 (Bladder)	clone 439-9B	Basal

Table 3.

Primary antibodies for prostate and bladder epithelial cultures [38, 40].

Method	Mechanism	Advantages	Disadvantages
Chemical Method	By using EDTA or EGTA it binds with cations and disrupt the intracellular bonds [42]	Easy and cost saving	It does not adequately dissociate all types of tissue
Mechanical Method	Cutting, scratching, the tissues in to small pieces in order to separate the cells and wash it with gentle agitation [42]	It is a rapid technique works best for loosely associated tissue. Correct temperature should be maintained for enzyme.	Decreases in the surviving capacity of the cell, incision of scissor, scalpel for cutting, scratching can damage the cell.
Enzymatic Method	Enzymes to cut or digest tissue pieces in free cells. Combination of enzyme also can be used; Ex.- Collagenase, Trypsin, Hyluronidase [43].	It has great specificity with specific enzymes	Enzyme dissociation can modify proteins on cell surface

Table 4.

Mechanisms for isolation of cells.

Although this is first step in primary culture, there is still no standardized protocol for this. There is a variety of options available. Tissue has to be mechanically minced from autoclaved scalpel or scissor; if tissue is measuring from 1 to 20 grams semi-automated dissociator can be used. Manual method has to be done in ice cold PBS (Phosphate-buffered saline). Commercially available formulation showed 10% increased viability compare to collagenase I, II, IV. (Table 5) [33]. In another study mechanical and enzymatic method has been used. In mechanical method, they used lacerate and scalpels and in enzymatic method collagenase type I and hyaluronidase type I enzymes with medium agitation at 37°C for 18 hours was used [34]. EDTA (Ethylenediaminetetraacetic acid)/Trypsin mixture used with 5 minutes of incubation in 37°C degree for prostate tissue [35]. Both the mechanisms, mechanical disaggregation with disposable disaggregator and enzymatic by collagenase and trypsin used for prostate tissue [36, 37]. In some cases, trypsin/EDTA 1:5 solution and incubation for 15 minutes for bladder tissue was used. In some studies for dissociation of bladder tissue 1:1 collagenase II and dispase enzymes are used at 37°C for 12 hours [40, 41, 42, 43]. Also there is a need to monitor tissue digestion process for every 2 hours by gently shaking the digestion mixture by checking the viability of cells under the microscope [50, 51, 52, 53].

Sl. No.	Components	Function
1	Buffering systems; Natural buffer system Chemical buffers system	CO2 balances the pH,5 to 10% Co2 incubation, non-toxic and cost effective [44] HEPES, buffering capacity7.2–7.4, in high concentration it is toxic, costly [45, 46]
2	Inorganic salt	Maintains osmotic balance and membrane potential by providing sodium, potassium, and calcium ions [47]
3	Amino acids	Require for proliferation of cells and provides nitrogen Ex. L-glutamine provides NAD,NADPH and serve as secondary source of energy in metabolism [48].
4	Carbohydrates	Carbohydrates are major source of energy, most of the media use glucose and Galactose.
5	Proteins and peptides	Major proteins and peptides in media are albumin, fibronectin and tranferrin. Albumin helps to remove toxic substances from cell culture media
6	Fatty acids and lipids	Particularly added when serum free media is used [44].
7	Vitamins	Important for cell growth and proliferation as cells cannot produce sufficient amount, need to provide through culture media [44].
8	Trace elements	Copper, Zink, Selenium required in trace amount for proper growth and many biological process
9	Antibiotics	Controls growth of bacteria and fungi ex.Penicillin, Streptomycin, fluconazole [49].
10	Serum in media	Serum is a complex mixture provides all above elements. Ex. Fetal and Calf bovine serum [44].

Table 5.

Basic components of media and their functions.

4. Tissue processing

After digestion, cells are strained by strainer to separate the debris from it. Then, cells micro clumps are washed with PBS or HBSS twice or tricefollowed by centrifugation [54]. Cell pellet collected from centrifugation is suspended in 2 ml of culture media. Count the viable cell by hemocytometer or by tryptophan dye exclusion method [55]. Cell viability also can be measured by the intracellular adenosine triphosphate levels which are commercially available kit [56]. Immunohistochemistry and immunofluorescence techniques used to localize, identify and quantitate the cells based on cell surface marker [57].

5. Culture media

Cell pellets collected from centrifugation has to be placed in micro well plate or flask that contains culture media. It provides artificial environment for cell to grow. Basic requirement of culture media are controlled temperature, substrate to attach cell, growth medium and incubator to maintain pH [45]. Main step in culture is to choose culture media. It generally composed of amino acid, vitamins, inorganic salts, glucose, hormones, growth factor, and attachment factor which provides energy and helps to complete the cell cycle. Commercially available cell media for primary epithelialcancer cells are less effective compare to tissue specific primary cell media prepared in lab (Table 6) [46, 47, 48, 49].

SL No.	Cell type	Components of media
1.	Fibroblast cell culture [28]	DMEM media with 7.2 pH + Fetal calf serum(FCS) + 100 U/ml penicillin,100 μg/ml streptomycin +1% amphotericin B added in culture media.culture plate incubated at 37°C a humidified chamber of 95% air and 5% CO2
2.	Prostate cancer (Bone metastatic variant) [29]	DMEM Glutamax +4.5 g/L D-Glucose with pyruvate +10%FCII +1% penicillin–streptomycin+37°C with 5% CO2.
3.	Bladder cancer (Epithelial cells) [58]	EMEM (ATCC) +10% FCS +1% penicillin + streptomycin + humidified incubator at 37°C with 5% CO2.
4.	Prostate cancer (Epithelial cells) [59]	KSFM medium+ + 25 mM HEPES +1% penicillin + streptomycin +0.5 mg/mL fungizone +100 mg/mL gentamicin +37°C, 5% CO 2 humidified incubation.
5.	Epithelial cells [40]	serum-free RPMI 1640 without phenol red+ penicillin 100 IU/mL+ streptomycin 100 μg/mL+ metronidazole 1 μg/mL+ amphotericin B 2.5 μg/mL + gentamicin 20 μg/mL + 37°C and 5% CO2 for 6 days.
6.	Bladder cancer (Urothelial cells) [50]	Glutamine + insulin + Phosphoethanolamine+ ethanolamine + hydrocortisone+ transferrin + EGF + BPHE+5% FBS +5% CO2 at 37°C
7.	Prostate cancer (Epithelial cells) [60]	DMEM + Glucose +100 U/ml penicillin +100 mg/ml streptomycin sulfate+0.29 mg/ml glutamine + Euroclone
8.	Prostate cancer (Epithelial cells) [61]	DEME /Ham’s (1:1) + BSA (0.01%) + FAS (2%) + Epidermal growth factor (10 ng/mL) + (insulin-transferring-selenium-1%) + hydrocortison (0.5 μg/mL) + Tryiodotyronone(1 nM) + phosphpethenolamine(0.1 mM) + choleratoxin (50 ng/mL) + fibronectin(100 ng/mL) + futine(20 μg/mL) + penicillin/streptomycin (100 U/mL,100 μg/mL) + R1881(0.1 nM).

Table 6.

Components of media from different studies.

Choice of culture media is very important to get significant result in experiment. Selection of media completely depends upon type of cell, purpose and resource [62]. As primary culture provides valuable research data, preparation of quality culture media is required, or to avoid limitation (cell number) of primary cell culture, commercially produced medias are available (Table 7) [58, 59, 60, 61].

Sl.No.	Name of media	Supplier
1.	Human Endothelial-SFM	Life Technologies
2.	Endothelial Basal Media	Sigma Aldrich
3.	EndoGRO-LS Complete Media Kit	MilliporeSigma
4.	HUVEC Basal Medium CB HUVEC	AllCells
5.	Endothelial Cell Medium	ScienCell
6.	Epithelial cell medium	ScienCell
7.	EpiGRO primary epithelial cells	MilliporeSigma
8.	RPMI 1640	Sigma Aldrich
9.	αMEM	ThermoFischer
10.	k- SFM	ThermoFischer

Table 7.

Commercially available media for epithelial cells [44, 63].

It is very important to maintain cell viability after isolation process which is totally depends on skillful handling and culture conditions. The culture condition will differ depending on the cell type. Cell growth has to be observed till 11 or 12 days. Additional extra media, Fetal Bovine Serum (FBS) and antibiotics need to be provided to avoid contamination. Culture media has to be changed between 2 and 3 days [60]. Initially apoptosis is 5% from 0 to 1 day but as days will pass apoptosis rate will increases from 7 to 14 days. But functional validity of benign and prostate cancer cells was 5 days after confirming it with histochemically, biochemical and by immunohistochemical assay [63]. Use of serum free culture media with low calcium condition increases the longevity of the cell. Cryopreservation (Preservation of structurally intact cells) can be achieved by adding 10% FBS (Fetal bovine serum) and 10% DMSO (Dimethyl sulfoxide) in 80% confluence primary cell culture [64].

Each day morphological changes have to benoted. Normal cells get counted every day and cancer cells get counted every 2 days [65]. Cell viability is determined by trypsin blue dye, equal volume of PBS and trypsin blue dye allowed to sit on cells for few minutes then to count the cells samples are loaded on hemocytometer, cells scored as leaving or dead based on uptake of tryptophan blue dye [66]. Once confluence reaches to 80 to 90% it has to get counted by Neubauer camera at 1:2 dilution with tryptophan dye exclusion, MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) can be used to determine cell viability [67]. Cell growth curve can be plotted from the graph to check the time when the cell viability increases in the culture. Once cell get cultured properly depending upon need of investigator, cells can be passaged and characterization of the cells can be done.

6. Cancer cell lines

Most established prostate cancer cell lines namely PC-3, DU145 (Duke University 145) and LNCaP (Lymph Node Carcinoma of the Prostate) developed in the 1970s and 1980s are in the majority of the published studies [68, 69, 70]. T24 cells (cell line from transitional cell carcinoma of the bladder) are exemplified for bladder cancer research [71]. These immortalized cancer cell lines are not always predictive of the real cancer behavior for the preclinical studies as these cells are adapted to 2D monolayer culture conditions [72, 73].

7. Cell culture models

Traditionally animal models were commonly employed for carrying out study of different types of cancer for the past three decades [74]. These animal studies have many drawbacks including lack of high-throughput drug screening, longer time consumption to conduct tests and ethical controversies concerning animal testing. Cell culture is the most widely used alternative to animal studies and cell culture techniques can broadly be classified into 2D and 3D methods [75]. The potentialities of primary cancer cell models’ cultures in preclinical studies for cancer research and drug discovery has amplified over the past few years. Primary cell cultures provide a good model system to understand normal and malignant biological activities. Carcinogenesis-related behavior such as apoptosis, proliferation, adhesion, differentiation, migration, senescence, invasion, angiogenesis, and other metabolic pathways have been studied in recent years. One of the major advantages is that the heterogeneity of cell populations composing a primary culture mimics the tumor microenvironment, crosstalk, and interactions between malignant and healthy cells, neither of which is possible with cell lines [76].

Most studies have shown that the cellular responses to drug treatments in 3D cell culture are significant and more similar to that of in vivo architecture when compared to 2Dcell culture. One of the most improved successful assays using 3D culture for cell-based screening in the early phase of drug discovery is cancer cell viability assessment. This assay is particularly useful to test the cytotoxic effects of compounds that may lead to cell death. It plays an essential role in checking how many cells are viable at the end of each experiment. Cell viability assay is closely followed by cell proliferation, cell migration and then cell signaling assays [77, 78, 79].

Currently a number of anticancer drugs belonging to different classes chemically are available. To be used as a potential anticancer agent, the testing compounds need to inhibit the growth and proliferation of cancer cell lines. This will further inhibit the signaling pathways by knocking in or knocking out a candidate gene thereby stopping the progression of tumor to fatal stages. For instance, antiproliferative investigations were performed on prostate cancer cell line DU-145 in vitro and in vivo using salvia miltiorrhizabunge [80]. Another example to justify this concept will be a study performed on PC-3 cell line. Generally, cancer cells express higher amount of Transferrin Receptors (TfR) for an increased uptake of iron in relative to normal cells. This higher amount of intracellular free iron is required for the growth and proliferation of cancer cells. Anhydrodihydroartemisinin (ADHA) was used to inhibit PC-3 cell lines through caspase-dependent pathway [81].

8. Applications: cell culture in drug discovery and screening

8.1 Drug candidate identification

Often the rate-limiting step in preclinical drug discovery is the target identification and validation step. 3D cell cultures have the potential to discover the molecular perturbations governing carcinogenesis and to accelerate target identification and validation, given that the gene expression patterns found in 3D models are relative to in vivo, when compared to 2D monolayer models [81]. For instance, a study reported that CXCR7 (C-X-C chemokine receptor type 7) and CXCR4 (C-X-C chemokine receptor type 4) were co-expressed in LNCaP, DU145 and PC3 cell lines in 2D culture. A marked up-regulation of both receptors was observed in PC3 cells when cultured in 3D using Matrigel suggesting that inhibition of CXCR7/CXCR4 may assist in controlling prostate tumor growth and subsequent progression [82].

8.2 Toxicity profiling

Cultured cancer cells are powerful in assessing drug-induced toxicity and to determine suitable drugs and methods for selectively destroying different types of cancer. It is useful to investigate effects of drug responses on metabolic signaling pathways or candidate genes conceding drug screening practices with impressive progress in the last decade. A study investigated features such as vascularization and perfusion of antineoplastic drugs on human T24 bladder cancer [83]. It allowed in the understanding of basic paracrine signaling mechanisms that regulates tissue homeostasis, development of new methods for urinary bladder reconstruction and tissue engineering, and generation of models of malignant and benign diseases. This study suggested that the use of 3D urinary bladder cultures could be a possible approach in clinical practice to select for the best antineoplastic drug for each patient and to investigate the effect of drug combinations or new antineoplastic drugs [84, 85]. The below (Figure 2) suggests how assay-guided treatment can be useful in choosing the best active drug for an individual patient.

Figure 2.

Assay-guided treatment choosing the best active drug for an individual patient.

8.3 Testing anticancer activities

By far the most useful in vitro model which is used to analyze the anticancer activity is Cell culture. Treatments including radiotherapy, chemotherapy, hormone therapy, novel and experimental therapies can be evaluated. Extracts of plants can also be utilized to check for anti-cancer behaviors such anti-inflammatory, destabilized membranes through which invasion and migration can occur. For instance, leaf extracts of Leea indica were used to study in– vitro antioxidant and anticancer activity on DU-145 and PC-3 human prostate cancer cell lines [86]. An example of drug combination is the synergistic effect of cisplatin and sunitnib malate – based chemotherapy on T24, 5637, and HT1376 human urinary bladder cancer cell lines [87].

9. Conclusion

Primary cell cultures have its application in various fields like toxicology, virology, drug screening, genetic engineering, gene therapy, genetic counseling, cancer research but main important application is model system. It provides best model system for studying basic cell biology and biochemistry, effects of disease-causing agent and cell, effect of drugs on cell, process which triggers aging and apoptosis. Primary cell culture represents excellent model for transitional preclinical experiments to understand cancer in in-vitro system. Primary cell culture acts as gold standard for cell line experiments because it provides broader spectrum of cell types from greater number of patients to be studied without any induction of artificial genetic mutation and it also maintains same phenotype throughout the culture. It involves both, clinician and researcher in the culture, it helps in understanding the drawback of treatments and lack in laboratory methodology and hence it is possible to overcome from it.

There are many models in cancer research; each model has different potentialities and inadequacy. In primary cell culture, complexity arises due to poor tissue quality, collection and inappropriate culturing may decrease the cell viability. Hence management of primary cell culture is difficult. But to overcome from these difficulties proper collection with the help of pathologist and selection of proper isolation method and culture media based on tissue type can help to increase the cell viability. Considering the current clinical system towards precision medicine, patient derived cancer models are powerful epitome in cancer research. Nowadays 3D model system is emerging system. Primary cell culture can help to model 3D culture, in future technological perspectives like 3D culture can replace the in vivo model system. In conclusion, this chapter reviews several aspects of primary cell culture, provides overview on selection of tissues, different methods of isolation, culturing media and management of cells after culture. It summarizes the wide range of studies to improve the understanding of prostate and bladder cancer preclinical treatment processes.

Conflict of interest

The authors declare conflict of interest as none.

Author’s contributions

KK & SIP: Worked on collection of data and drafted the chapter, AAA: Worked on the draft of cell lines images and edited, SCG: Developed the study designedand edited the chapter, RBN & MBH: Collected the literature and guided throughout the study. All the authors reviewed and approved the chapter.

Funding

Nil.

Abbreviations

ADHA	Anhydrodihydroartemisinin
BPHE	Brazed Plate Heat Exchangers
BSA	Bovine Serum Albumin
CD cell markers	Cluster of Differentiation cell markers
DMEM	Dulbecco’s Modified Eagle Medium
DMSO	Dimethyl sulfoxide
DU145	Duke University 145
EDTA	Ethylenediaminetetraacetic acid
EGTA	Ethylene glycol tetraacetic acid
EMEM	Eagle’s Minimum Essential Medium
FBS	Fetal Bovine Serum
FCS	Fetal Calf Serum
FGF	Fibroblast Growth Factors
HBBS	Hanks’ balanced salt solution
HEPES	Hydroxyethylpiperazineethanesulfonic acid
IgMI	Imunoglobuline M Imunostain
KSFM	Keratinocyte Serum Free Medium
LNCaP	Lymph Node Carcinoma of the Prostate
PBS	Phosphate buffer saline
RPMI 1640	Roswell Park Memorial Institute (RPMI) media
TfR	Transferrin Receptors