**2.1. 2D skin cell cultures**

maintained in controlled environments and under controlled temperatures for cells to maintain their ability to survive and to grow [3]. For this reason, a medium for the cells to obtain

Cells obtained directly from living tissues and then cultured are referred to as primary cell cultures [4]. These cells have a short time frame of survival and scientific use [4]. Cells that have been modified to survive indefinitely with continuous cell division are referred to as either continuous cell lines [1] or immortalized cell lines [2]. Cancer cells are an example of the type of cells that may be used to create cell lines due to their genetic mutations and ability

The very first cell cultures developed were of human skin which allowed for a better understanding of the physiological functions of the skin [5]. Cell cultures are available today in twodimensional (2D) and three-dimensional (3D) forms. Cell cultures that grow in controlled flat environments, such as a Petri dish, are 2D cell cultures [6]. Cell cultures that are 3D, however, combine and shape cells into a 3D form using a surrounding medium or specialized conditions to help maintain a 3D figure [7]. *In vitro* skin cell cultures developed initially were primarily 2D with keratinocytes as the primary cell types [8, 9]. In the epidermal layer, the main cell types are keratinocytes, while fibroblasts are the main cell types in the dermal layer of the skin [10]. It is thus evident that an *in vitro* model consisting of both keratinocytes and fibroblasts is required to better mimic the physiological functions of the human skin, especially with relation to the wound healing properties of the skin [5]. There are therefore ongoing efforts to develop various *in vitro* skin models that more closely resemble the skin's physiological functions and that will allow for testing of dermatological products [8]. To have a better understanding of the type of cell cultures used for testing dermatological products, this chapter aims to outline the differences between 2D and 3D skin cell cultures while considering the advantages and disadvantages of each culture. Different types of cell cultures used for wound healing and for inflammatory skin conditions such as psoriasis will also be

2D cell cultures have been the main type of cell cultures used by scientists in various fields for decades [6]. As explained in the introduction, cell cultures that grow in controlled flat environments, such as a Petri dish, are 2D cell cultures [6]. To maintain cells outside of an organism's body, it is important to have the correct equipment and supplies to ensure the provision of an environment in which cells can survive and divide [2, 3]. Equipment involved typically includes a biosafety hood for maintaining the safety of cultures and of the individuals working with the cultures, a cell incubator for correct storage with a temperature of 37°C

Besides having the correct equipment for adequate handling and storage of cell cultures, the culture medium or environment in which cell culture grow is also important. The cell culture

dishes for placement of cells, amongst other required equipment [2, 3].

, a refrigerator for culture media and other supplies, and flasks or Petri

the required nutrients for their growth is essential [2].

to divide continuously [2].

4 Cell Culture

discussed.

**2. 2D cell cultures**

and 5% levels of CO2

The very first cell cultures developed were derived from human skin and were of primarily keratinocytes [5, 8, 9]. As the largest organ in the human body, [14] the skin consists of the epidermal and dermal layers [14, 15]. In the epidermal layer, the main cell types are keratinocytes, while fibroblasts are the main cell types in the dermal layer of the skin [10]. Initially, 2D skin cell cultures consisted primarily of keratinocytes [8, 9]. It was however determined that fibroblasts were essential for the growth of keratinocyte cultures [16] as fibroblasts are the cells responsible for secreting substances of the extracellular matrix such as collagen [17]. The primary fibroblast cell line, cultured by George Todardo and Howard Green, is known as the 3T3 cell line and was named based on its culturing method of transferring cultures every 3 days [16, 18]. The 3T3 cell line is typically used as a feeder layer in keratinocyte cultures [18]. HaCaT cells are a cell line of keratinocytes [19].

Prior to animal testing and clinical trials, traditionally, when testing pharmaceutical ingredients, the first step has been to test *in vitro* using 2D cell cultures [7]. This has however provided limitations with respect to testing as the results obtained from *in vitro* 2D cell culture studies have not translated to *in vivo* studies [7]. For this reason, *in vitro* 3D models have become increasingly popular as they are considered to more closely resemble *in vivo* processes [11, 20].
