Literature Review Geometric Control Of Cell Life Death Biology Essay

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This journal focused on the research of controlling mammalian cell growth in vivo and determined if the cell shape or integrin αvβ3 antagonists binding can control apoptosis. The local differentials in cell growth and viability that drove morphogenesis in complex tissues were controlled through modulation of cell binding to extracellular matrix (ECM). Human and bovine capillary endothelial cells were grown on ECM coated adhesive islands of varying size or on ECM coated micro beads over a range of different sizes. The ECM used in the research was fibronectin (FN).

The research focused on the efficiency of two models of beads and island. Micro carrier beads of different sizes coated with FN were used for cell growth. Soluble integrin would induce apoptosis in cell culture, but death of cells can be prevented by suspending the cells with immobilized integrins or by inhibiting tyrosine phosphatases. Cell death in vivo was caused by ECM dissolution that is supported by a possibility that cell may grow on large micro beads (>100μm) whilst they rapidly die when attached to small (4.5μm) ECM coated beads. The capillary endothelial cells were cultured in suspension in absence of any micro beads. 60% of the cells entered the dead phase after 24 hours. The death of cells were tested, after 24 hours, with different size of beads planar and the apoptotic index was quantified by measuring the percentage of cells exhibiting positive TUNEL and DAPI staining. This could result the trend of death rate with the size of beads. Two samples of cells attached to different size beads, from a suspended culture or attached to a dish, were taken. The highest rate of cell death was observed from the suspended, 60% of cell died in this planar. Micro scale patterning technique was use to fabricate planar adhesive islands. The death rate in attached planar was very small as compared to the suspended culture. The death rate was approximately 10% for beads at size of 25μm, and lower for size of 45μm. A significant increased in growth rate was observed at size of 10μm. These results confirmed that the cell attachment on ECM mediated by integrin receptors was important for cell survival.

Using a scanning electron microscope (SEM), it was observed that the 10μm beads were fully engulfed by the cells in 4 hours. But beads of 25μm, cells adhered and remained flattened. It was observed that cell spreading was increased on homogeneous high density coating of FN which also leads to the cell survival and growth, yet, cell ECM also increased. Single cells bodies were comparable to focal adhesion on islands of gap 10μm or 20μm and stretched towards the closely placed island. The cells grown on 10μm became more rounded and the apoptosis rate was increased to around 20% for 10μm and 10% for 20μm. By changing the distance between the islands, cell ECM could be altered significantly. This showed that the death rate was higher with a larger island gap and results confirmed that the extension of cell spreading played a significant role in cell death and/or survival.

DNA synthesis was directly related to cell area and apoptosis was switched off in the event of cell spreading. Cellular proliferation and apoptosis was determined under varying growth condition. When cells were grown on different sized, square-shaped FN coated islands, apoptosis declined and DNA synthesis increased with size ranging from 75 to 3000µm2. Death rate for different sized islands were measured by TUNEL staining and the results were plotted and showed that a larger surface area of the island possessed a better condition for cell spreading and growth This was also observed that by increasing cell spreading on a homogenous FN coated would lead to cell growth when the total area of cell to ECM attachment were kept constant. Under this conditions growth conditions, DNA synthesis increased and apoptosis decreased with increased in cell spreading. Using substrates coated with specific antibodies to various integrins, the anti-apoptotic effect of spreading was greater with selected integrin αvβ3. By reducing integrin αvβ3 binding, cell death could be prevented in vivo and apoptosis could be prevented by attachment of cells to immobilized cell bodies such as β1 integrins. β1 and β3 integrins could assist cell binding in FN coated medium. When unpatterned substrate on 20μm circular beads with anti-αvβ3 and β1 integrins were used, apoptosis decreased up to 60% in suspended cells but when cells spread on unpatterned islands the survival rate was high. When spreading was restricted, cells adhered to β1 went to apoptosis at a higher rate as compared to cells adhered to integrin αvβ3. As a result, change in the microenvironment of cell could promote cell death. Cells in the course of morphogenesis growing and dying co-exist and living cells could result in cell deformation. A progressive loss of shape-dependent could lead to cell survival, but it may cause neo-plastic disorganization of tissue architecture.

Criticism discussion

The research was focused on the study of cell adhesion in growing mammalian cells. Experiments were carried out to investigate shape dependent cells on apoptosis. Results from the experiment showed that adhesion could encourage cell growth, and specific adhesion types could possibly control the death and growth of cells. Micro carrier beads of different sizes were used with similar adhesive coating with a constant area of ECM.

60% of the cells, when cultured in suspension form, went into cell death program. The authors observed that if the distance between islands was varied, apoptosis could be controlled. From the experiments, integrin receptors were significant for cell growth. From figure 3D, growth and apoptosis rate were shown. The reason for this was not clear and as a result of this, a few possibilities were raised. For example, cells could be having a high apoptosis rate and as well as a high death rate. If this happened, cells then used DNA synthesis to form new cells using the remaining cells that survived. The surface area of ECM could be made constant with varying cell spead and this would result in decreased rate of apoptosis. When cells were cultured on smaller circular beads, they took the shape of island and as a result, their apoptosis rate was increased. For cells with high apoptosis rate, the pattern was suitable for cell growth. The rate of apoptosis could be decreased using specific substrate coating. The results showed that shape of cells were concluded to be the critical factor in determining cell growth, life and death.

Throughout the journal, there were no actual facts describing the death of cells. When cells were removed from its original habitat, factors such as lighting conditions, surrounding temperature, source of energy available, surrounding thermodynamics or even nutrients available in the culture could affect cell growth and death.

Multiple cell growth was observed as mentioned on page 1427. A cell could be attached to several islands and the gap between those islands could be twice of the diameter of the islands. Cells could be attached onto many different kinds of adhesive pattern designs such as scaffold. The gap between the islands was determined by the author. The author failed to mention if distance between gaps were altered under the same or different operating conditions. If additional experiments were carried out under random conditions, it would give a clearer perspective, such as the varying gaps on the growth and death of cells.

Suggestions for future research

The experiment carried out on the rate of cell growth and death or apoptosis should be measured with respect to time. If the length of time were measured, cell growth or death could be better observed and analysed, and also errors which occurred during the course of the experiment could be eliminated. The distance between the gaps of the adhesive islands should be analysed further, for example if increasing distance between gaps would assist in cell growth.

Further research on ECM coated materials would greatly promote cell growth and provide a better control over cell growth and death. The ECM coated materials could also encourage cells to attach and spread on micro beads or islands which would result in improved method of controlling cell spreading. Furthermore, if apoptosis were forced on surviving cells, then these cells would remove itself from cell adhesion.

Research could be done on the micro carrier beads design which would help further understand the basic idea of processes that could then be scaled up for large scale bioreactor. The chemical inhibiters for apoptosis like integrin αvβ3 and anti-β1 extended cell culture could help in genetic engineering and tissue engineering.

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