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Fibroblasts are the cells synthesized in extracellular matrix and collagen. They are most abundant in the connective tissues and play vital role in wound healing. Cell proliferation in fibroblast can be studied during the process of mitosis.
Mitosis is the process of cell division in eukaryotes in which the chromosomes present in the nucleus of a cell divides resulting in the formation of two identical nuclei (Irwin R.,Wick SM, 2008). This process is generally followed by cytokinesis, which leads to division of organelles, cytoplasm, and cell membrane into two parts. Mitosis and cytokinesis together constitute the M Phase or Mitotic phase of cell cycle. The major aim of mitosis is the division of parent cell's genome into two daughter cells which are genetically identical to the parent cell. Hence the parent cell has to make a copy of each chromosome prior to mitosis in S phase of interphase.
Interphase is the the period of cell cycle prior to mitotic phase when the preparation for mitosis takes place (Blow J., Tanaka T, 2005). Interphase is a much longer phase of cell cycle which is divided into three phases namely G1 phase(First gap), S phase (synthesis phase) and G2 phase (second gap). Cell growth by production of proteins and cytoplasmic organelles takes place during these three phases of interphase. Interphase is followed by prophase during which loosely bundled coil of chromatin condenses to form chromosome. Due to the duplication of genetic material in S phase, each chromosome has two chromatids in prophase. These two sister chromatids are joined together at centromere by a specialized cohesion complex. Centrosome (each of which is made up of two centriols) coordinates the microtubules of the cell. Spindle fibre formation takes place towards the end of prophase. Prophase is followed by prometaphase during which nuclear envelope disassembles and microtubules invade the nuclear space. The chromosome forms kinetochores at centromere, which remains attached at chromatid. The microtubules attach at these kinetochores. The connection of microtubule with kinetochore leads to the activation ofATP mediated motor system, which ultimately leads to the separation of the two chromatids of the chromosome. ( Maiato H , Deluca J., salmon E., Earnshaw W, 2004)
During metaphase the spindle fibres are developed completely and chromatids are positioned at the equatorial plane of the cell. Spindle fibres are properly attached to the centromere and kinetochore; and the contraction of the spindles makes the chromatids to separate properly.
In anaphase, chromatids separate due to the cleavage of the binding protein. The sister chromatids are then pulled apart by shortening of kinetochore microtubules. After that , the non kinetochore microtubules gets elongated, pulls the centromeres apart to the opposite ends of the cell. Thus, by the end of anaphase , the cell gets divided into two populations of identical copies of genetic material.
Fig. 1: Prophase stage Fig.2: Metaphase stage
Fig. 3. Anaphase stage
[ chromosome in blue colour, spindles in green, and actin in red colour]
Once the chromosomes reach the opposite poles of the spindle, telophase begins. At this stage, spindle disappears, the chromosomes extend by uncoiling, the nucleoli slowly appear again, and the nuclear envelope is again formed. Finally, by the process of cytokinesis, the entire cell divides into two daughter cells which enter the phase of interphase, thus completing one circle of cell cycle.
Study of cell proliferation in human derived fibroblast involves the technique of sub culturing in which the fibroblast cell lines are maintained by repeated transfer of them from one culture medium to another, thus avoiding the potential scarcity of the nutrients. This experiment involves the culturing of the provided cell lines with and without serum, followed by the treatment of some of the sub cultured cell lines with colchicines which prevents the microtubule formation and thus arrest the metaphase stage of cell.
In order to demonstrate the cell proliferation of human derived fibroblast, a confluent monolayer of the cell was taken from an old medium in a tissue culture flask , and the old medium was replaced with 5 ml of sterile phosphate buffered saline (PBS). After trypsinization of the cells with 3 ml of 0.25% of trypsin , 2 ml of medium was added to this preparation and numbers of cells present in this suspension were counted by haemocytometry. After that, 0.2 ml of this suspension was then added on wells 1, 2 , 4 and 5 which were already incorporated with collagen-coated cover slips. This preparation was then incubated at 37 degree Celsius. After 48 hours, colchicine was added to well 2 and 5. Similarly, after 54 hours, medium was removed from each well, and rinsed with PBS. Finally, 4% buffered formalin was added to the wells and incubated at 4 degree Celsius. On the next day, each coverslip was rinsed with distilled water and mounted on glass slide with one drop of Vectashield mountant. Finally, the slides were examined under fluorescence microscope and the images were recorded.
1 cell was seen in the area of 1 mm2 and depth 0.1 mm
i.e. 1 cell is present in the volume of 0.1 mm3
i.e. 1x 10 cells are present in the volume of 0.1x10 mm3( or 1 mm3 or 1 micro litre)
i.e. 10x103 cells (or 104 cells) are present in the volume of 1 x103 micro litre (or 1 ml)
Hence 104 cells are present in 1 ml of cell suspension.
Human derived fibroblast cells from tissue culture
Fig 4: Image of human derived fibroblast taken from culture medium containing serum (well 4), under 40x magnification of fluorescent microscope
In this experiment, fibroblast cells grown in medium without serum show poor differentiation than those containing serum in the medium. Serum is a good source of hormones, growth factors and transport or binding proteins which provide nutrients or hormones to the cell (Perez-Infante et al., 1986). In addition, serum contains some attachment factors like fibronectin and serum spreading factor which promotes the attachment of the cell to the substratum. Serum also plays a vital role in detoxifying and stabilizing the culture environment by binding with free fat soluble vitamins and steroid hormones. Additionally, it detoxifies the heavy metals and reactive organic compounds present in the medium (Mather et al, 1986). Due to these attributes of serum, if any culture medium lacks the incorporation of serum, there might be the consequences involving the accumulation of toxic substances or the scarcity of hormones or growth factors inside the culture medium, resulting in poor cell differentiation and growth.
In above experiment, when colchicine was added to the culture medium, mitotically dividing cells were arrested at the metaphase. The metaphase stage of mitotically dividing cell shows some characteristic structural features like equatorial positioning of the chromatids, and the appearance of spindle fibres. Apart from the above mentioned techniques, metaphase can be experimentally studied by another technique called immunohistochemistry where proteins like beta-tubulin, or NuMA (Nuclear Mitotic apparatus ) protein can be used as markers for the metaphase stage of cell division in fibroblasts.
Immunohistochemistry is the technique of localization of antigens (or proteins) in a tissue by using labelled antibodies as specific reagent through an antigen - antibody reaction that is visualized by a fluorescent dye under microscope. Figure 5 shows the theory of the immunohistochemistry technique involved in the study of the metaphase stage of human derived fibroblast. Class III beta tubulin is the component of spindle seen at metaphase of normal fibroblasts. Two different Tuj-1 antibodies specific for class III beta tubulin are used to localize the fibroblast cells at metaphase. An intense immunoreactions can be seen during the metaphase stage of cell division when the microtubules are connected to the kinetochores of the chromatids.(Jouhilahti EM,Peltonen S,Peltonen J.,2008) Similarly, NuMA protein is another marker for metaphase which shows specific attachment sites on metaphase chromosomes and mitotic spindle poles. (Van Ness, Pettijohn and Klug 1983). Thus, apart from sub culture technique, metaphase stage of the human derived fibroblast can be studied by immunohistochemical technique where biomarkers like Class III beta tubulin or NuMA protein are used as the indicators of metaphase.
When the cell suspension of Human derived fibroblast (containing 104 cells per ml) was subjected under culture condition with and without the incorporation of serum, degree of growth and cell proliferation was found to be significantly different in different media. Cells incorporated with serum showed marked cell proliferation in comparison to those not incorporated with serum. Similarly, cells treated with colchicine showed the arrest of the cell cycle at metaphase.