**2.** *In vitro* **applications of 2D culture**

#### **2.1** *In vitro* **cytotoxicity assays and tissue-engineered tissue models**

Cytotoxicity assays are commonly used for *in vitro* toxicology and pharmacology studies for the screening the effect of chemicals and drugs on the cultured cells. There are different assays available for measuring cytotoxicity namely- the colony-forming assay and dye inclusion or exclusion such as neutral red and trypan blue assay is the most significantly used. Cytotoxicity assays can be broadly divided into the following categories– (i) Assays based on metabolism, (ii) Adenosine triphosphate (ATP) Bioluminescence Assay and (iii) Assays based on the release of enzymes.

Assays based on metabolism generally include the 3-(4,5-dimethythiazol2 yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and its alternatives such as 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium(MTS) and sulforhodamine B (SRB) assay. Due to rapid, quantitative, versatile, and highly reproducibility of MTT, it is widely used in largescale, anti-tumor drug-screening program. MTT is a quantitative colorimetric assay that quantifies the reduction of yellow tetrazolium dye by mitochondrial succinate dehydrogenase to purple insoluble formazan crystals by the NADPH dependent cellular oxidoreductase enzymes [12]. The crystals are dissolved in an appropriate solvent. The absorbance is then recorded using a spectrophotometer to analyze the cell viability wherein the crystals get accumulated in the viable cells due to their impermeability to the cell membrane.

ATP Bioluminescence Assay is used to measure the ATP level that is well regulated in the metabolically active live eukaryotic cells as compared to the dead cells wherein the ATP level falls due to the activity of ATPases. This assay includes a luciferase enzyme that utilizes energy from ATP that converts luciferin into oxyluciferin and thus produces luminescence. Therefore, luminescence could be used to measure the ATP level. Assays based on the release of enzymes are more significant as they measure the products released by the dead cells [13].

Assays based on the release of enzymes include Lactate dehydrogenase(LDH) leakage assay involving the formation of pyruvate from lactate in the presence of LDH with simultaneous reduction of NAD to NADH that alters the absorbance at 340 nm [14]. Research in cancer and cell biology is greatly dependent on *in vitro* assays and models. This help in understanding the various responses of the cultured cells when exposed to different conditions. Tissue-engineered *in vitro* tissue models serve as

an alternative to *in vivo* animal studies to study the physiology of various diseases. Example of tissue-engineered *in vitro* models includes Skeletal Muscle Models [15], blood–brain barrier model [16], aneurysm models [17] and the Pre-vascularized Human Vaginal Mucosa model [18].

### **2.2 Cell migration assay**

Cell migration is well known for its significant role in embryonic morphogenesis, cancer invasion and metastasis, immune responses, tissue formation, and angiogenesis [19]. Mainly, cell migration is of two types; single-cell migration and collective cell migration. Single-cell migration is regulated by cytoskeletal activity without cell-to-cell interactions with neighboring cells. This type of migration is important for embryonic development, immune response, and in the early stages of metastasis. On the other hand in the collective cell migration, the group of cells retains their cell to cell interactions as well as collective polarity. Wound healing assay or scratch assay is a 2D *in vitro* technique used to study collective cell migration. In this assay, a scratch is made on the confluent cell monolayer resulting in the formation of a gap or wound which is monitored by taking pictures of the migrating cells at regular intervals of time. These pictures are then used to measure the speed of wound closure and thus quantify migration. Live-cell imaging using Time-lapse microscopy can be used for a more detailed study of cell migration behavior [20]. In order to reduce the effect of cell proliferation on gap filling, the readings are taken for a time period of 24 hours but this may vary depending on the cell line.

Another assay involving the response of single cells to various chemo-attractants is the transwell assay or the Boyden Chamber assay. This assay can be used for both adherent and non-adherent cells wherein the cells are placed in a serum-free medium on one side of a porous membrane and analyzed on the basis of the cell's ability to migrate through the pores to the other side. Cell migration can be quantified by counting the cells that have traversed through the membrane towards the higher concentration of chemoattractant [21]. A drawback of this assay is visualizing the cells and their morphology while migrating through pores due to the transitive state of cells [22].

#### **2.3** *In vitro* **tumorigenicity assay**

Cancer is one of the most frightful diseases in both developing and developed countries and imparts a major health burden to the society. Tumorigenicity is the tendency of the cultured cells to form tumors. The two common *in vitro* tumorigenicity assays are - Colony-forming assay and Tumorsphere assay. The colony-forming assay is also referred to as clonogenic assay that analyzes the potentiality of a single cell to undergo a clonal expansion to form a colony composed of a minimum of 50 cells [23]. This assay is usually used to distinguish there productive viability of untreated cells from the cells that are treated with ionizing radiation or cytotoxic agents. It is also used to study the stemness and the clonogenicity of stem cells [24].

Colony forming assay is performed using the soft agar method. The basic steps involved in this assay are treating the cell monolayer in the flask, seeding the required number of cells on the agar and incubate for 1–3 weeks, fixing and staining the colonies and finally observing the colonies under the stereomicroscope [23]. Another *in vitro* tumorigenicity assay is tumorsphere assay which analyzes the potential of cancer stem cells (CSCs), a major cause of tumor initiation, progression, and recurrence

after treatment. This assay is carried out under non-adherent conditions and serumfree medium supplied with growth factors of choice leading to the proliferation of CSCs and formation of spheres whereas the non-CSCs undergo apoptosis due to loss of adherence and abundant nutrients [25].
