Lab Report

Analysis In the experiment, the scientist observed a series of cells, the first one being cheek cells. After swabbing the inside of their cheeks and preparing a slide, the scientists were able to see the cytoplasm, nucleus and cell membrane of this undyed cell. While observing these cells under 400X, they noticed that the cheek cells varied in shape, some being almost perfectly spherical, while others resembled an oval figure. Additionally, these cells appeared to be grouped up and messily stacked on top of one another.

Animal cells are the ‘cell factories for the production of complex biomolecules and antibodies for use as prophylactics, diagnostics or therapeutics’.Culturing of immortalized animal cell, also called cell lines is generally performed under define temperature, humidity and carbon dioxide flux in lab condition. Such in in-vitro culturing of animal cells may be performed under two different conditions viz. on solid surfaces (anchorage dependent cells) or in suspension (non-anchorage dependent cells).The choice of bioreactors and surface to be used for large scale cell culture is often decided on the basis of cell specific demands, engineering aspects and economic and regulatory considerations [Catapanoet al., 2009]. Generally, bioreactors

Coat a T75-cm2 culture flasks with 0.2% gelatin to provide better adhesion surface for mEFs.

Three wells where eliminated from the experiment, because a vehicle control was not needed for this experiment. Images where then taken of the 9 wells, with the use of phase-contrast microscope, these photos were labeled (T0). After the images where obtained, the well-plate was placed back into the tissue culture hood, and the wells where treated with different treatments, so the effects could be observed. In row “A” contained the HepG2 cells by themselves, no treatment was added. Row “B” contained the HepG2 cells and 4.5µl of Ceramide, which was labeled as the positive control, and finally in row “C” the wells contained our HepG2 cells and the compound Epigallocatechin Gallate. The well was then placed back into the cell culture incubator for 24 hours. Following the incubation, photos where taken using the same phase-contrast microscope and the same area of each scratch, these photos are labeled (Tf). The before and after photos of the scratch are then used to figure out the percent wound closure, which determines the cell migration. In order to calculate the percent closure, the following equation was used; Percent closure=width of Tf box/width of the T0

diH20 acted as a negative control in the experiment, all cells, both in intrafollicular zone and in interfollicular zone stained purple. Cells stained brown in the negative control indicate non-specific staining.

A hemocytometer is a glass slide, known for its feature of having parallel and perpendicular lines which are used to count the number of cells (Kazam et al., 1991). The formula for cell viability was established by counting the number of cells that did not have Trypan Blue over the total number of cells. The cell density was established by using the microscope and the hemocytometer to count four large squares and dividing it by the total volume of the squares. After this was finished, the media was replaced with a different with a different solution that contained 2% DMEM adult horse serum, 1% penicillin/streptomycin and 2% amino acids. These were used in order to allow for cell differentiation. Dulbecco’s Modified Eagle Medium(DMEM), is a nutrient mixture that contains vitamins, glucose and amino acids that assist cells in differentiating. The cells on the plates were viewed with an inverted microscope and were then placed in the incubator for four

They used those cells to coat sticky hydrogel beads, and then they partitioned these beads into small wells, each only seven millimetres across. Inside each well, the lung cells grew around the beads, which linked them and formed an evenly distributed three-dimensional pattern. To show that these tiny organoids mimicked the structure of actual lungs, the researchers compared the lab-grown tissues with real sections of human lung. When researchers added certain molecular factors to the 3D cultures, the lungs developed scars similar to those seen in people who have idiopathic pulmonary fibrosis, something that could not be accomplished using 2D cultures of these cells.

In Figure 1 an unstained check cells is shown clearly under a light microscope of a magnification of 100. According to Figure 1, the unstained check cell is not entirely visible under the microscope because no stains are used. Figure 1, shows some clusters of bubble-like structures, however can not be concluded that they’re check cells.

The cells were harvested, centrifuged at 250 x g for 10 min. Slides were prepared by centrifuging the cells using a Cytospin (Thermo Shandon, U.K). 250 μl of cell suspension was loaded in each block of cyto-funnel and centrifuged at 250 x g for 8 min. Two slides (two dots on one slide from each replicate culture) were prepared for each concentration. The slides were air dried, fixed in chilled methanol (90%) for 5 min, and stored at room temperature until stained (Figure 2.15).

(Chan & Leong, 2008). Biocompatibility is also an important factor. The material should be biologically compatible with the host tissue so it does not cause an immune or inflammatory response. (Pluta, Malina, & Sobczak-Kupiec, 2012). While the cells grow on the scaffold they generate a matrix around themselves, the scaffold provides structure and is eventually absorbed by the body; leaving the newly formed tissue to manage mechanical support. (Pluta, Malina, & Sobczak-Kupiec, 2012). The scaffold needs to be absorbed by the surrounding tissue at a rate compatible with the rate of tissue growth, so the new tissue can function independently and avoid the need for surgical removal. Lastly, to ensure functionality, the mechanical properties of the Scaffold should compatible with the tissue type. (Pluta, Malina, & Sobczak-Kupiec, 2012; Chan & Leong, 2008).

The extracellular matrix (ECM) is a feature of all multi cellular animals . It is a complex network of proteins secreted locally by cells via exocytosisalberts. The protein components that form the ECM include proteoglycans, glycoproteins such as fibronectin and fibres such as collagen. In addition a family of five matricellular proteins called thrombospondins are present.

Scanty squamous epithelial component (less than 8,000 well preserved and well visualized cells on conventional slides or less than 5,000 well preserved and well visualized cells on liquid-based preparations)

The rLaminin-521 in the previously coated plates were removed and replaced with fresh medium. For hESC culture, the MEF media was removed in place of the rLaminin-521. iPSC/hESC plates were refreshed with 1mL of new media, and the cells were manually passaged by lifting the colonies with a pipette tip. The cells were then evenly distributed among the wells and placed in the incubator.

The following media were sterilized by autoclaving at 121ºC for 20 min and initial pH was adjusted before sterilization using 1N NaOH or 1N HCl.

Cells are then centrifuged and re-suspended in sialidase and buffer and incubated to remove any sialic acid carbohydrates.