**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].

*Overview of Primary Cell Culture Models in Preclinical Research of Prostate and Bladder Cancer DOI: http://dx.doi.org/10.5772/intechopen.99493*

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 [33–37, 39–41]. After collection, tissue should be placed in sterile container which has HBSS (Hanks' balanced salt


#### **Table 1.**

*Biomarkers for Prostate cell culture.*

#### **Table 2.**

*Biomarkers for bladder cell culture.*


#### **Table 3.**

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


#### **Table 4.**

*Mechanisms for isolation of cells.*

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**).

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


*Overview of Primary Cell Culture Models in Preclinical Research of Prostate and Bladder Cancer DOI: http://dx.doi.org/10.5772/intechopen.99493*

#### **Table 5.**

*Basic components of media and their functions.*

(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 [40–43]. 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 [50–53].
