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CHAPTER 1 : INTRODUCTION

1.1 INTRODUCTION

Cell culture is a process by which the cells are removed from an animal or plant to a favorable artificial environment, generally outside their natural environment. It is a major and essential tool in molecular and cellular biology, and is progressively being used in order to provide accomplished model systems for normal physiology and biochemistry of cells study, drug screening, process of aging, study of the effects of drugs and toxic compounds of the cell other than to study the development of biological compounds (Antoni, Burckel, Josset, & Noel, 2015). In tissue engineering, cultures of dorsal root ganglia (DRG) are widely used and remained popular choices for basic neurophysiological investigations, for example in the study of molecular mechanisms of neuropathic and inflammatory pain in disorders of the peripheral nervous system as they are easy to be identified and isolated because of their comparably large size and simple geometry. In order to mimic the actual interactions that occur in the PNS, DRG have been used widely in co-culture system with Schwann cell (SC).

Dorsal root ganglion neurons (DRGs) are collections of sensory neuron cell somas located in the dorsal intervertebral foramen which are adjoining to the spinal cord, between the dorsal root and spinal nerve. They occupy both the peripheral nervous system (PNS) and central nervous system (CNS) which make them as an important role in transmitting information from the external environment to the spinal cord. It contains pseudonipolar neurons which are the afferent axons that transmit sensory information from the periphery to the CNS. Compared to the central nervous system, regeneration in PNS is possible. However, defective or insufficient peripheral nerve regeneration may contributes to despairing of the surgical patient.

In cell culture, one of the most important procedures is the isolation and cell harvesting which are the principle applications for enzymes in tissue culture research and cell biology studies influencing yield, viability and function of the cells. The goal of cell isolation procedure is to maximize the yield of functionally viable. One of the primary factors that can influence the outcome is the concentration of collagenase. Collagenases are enzymes that play an important role in breaking down and cleave the peptide bonds in the triple helical collagen molecule. The complex composition of connective tissue has to be broken down as tissues need to be dissociated in research or clinical situations, such as the preparation of suspensions of viable cells for metabolic studies, the isolation of pancreatic islets in diabetes research and the clinical treatment of necrotic tissue. Collagenases of the bacterium Clostridium histolyticum are of special interest among researchers as the structure and mode of action of this family of enzymes have been clarified.

1.2    PROBLEM STATEMENTS

Peripheral neuropathies are diseases of peripheral nervous system which can affect all of the components of the PNS, but the sensory neurons are often affected early and more severely as proven in the symptoms experienced by the patients which include sensory symptoms such as pain, paresthesias, loss of perception and gait imbalance. Peripheral neuropathies are common in predominantly young and working populations in which normal restoration of function are nearly impossible resulting in impaired hand sensation, reduced motor function and frequent pain and cold intolerance. This injuries often contributes to persistent impact to the patient and difficult to treat which will seriously affect their ability to perform daily activities and works. Numerous studies have revealed  that primary sensory neuron and its soma in the dorsal root ganglion (DRG) are essential sites for pathogenic functional changes leading to pain, including that which follows nerve trauma although neuropathic pain is associated by the alteration of the function in the spinal cord and brain (Hogan & D, 2003). For all these reasons, the ability of DRG to promote nerve regeneration has been a research area of intense focus. Furthermore, a massive body of evidence had proved the ability of mammalian DRG neurons to survive and regenerate in culture (Burkey, Hingtgen, & Vasko, n.d.).

Thus, bioengineering attempts in regeneration of DRG focused on creating a permissive environment and optimum requirement for DRG regeneration with no interruptions interface between the CNS and PNS. Moreover,  the isolation and cultivation of the dorsal root ganglion plays an important role in order to maximize the yield of functionally viable cell besides the ability to harvest large number of cells. Consequently, it is important to develop and modify a technique which can cuts down the time needed to produce the amount of cells needed in a short time, and one of the critical importance to the success of this technique was the incubation in collagenase which softened the DRG and allowed gentle dissociation. The choice of technique over another is crucial to understand the modifications that could lead to even better results and one of the factors is the concentration of the collagenase used to dissociate the tissue.

In this experiment, different concentrations of collagenase will be used to meet the optimum requirement in order to maximize the yield of functionally viable, dissociated cells. In order to use in research studies, development of new drugs, transplantation, and tissue engineering, viability and function of the cells is very important to be taken into account.

1.3      OBJECTIVES

1.3.1 General Objectives

To isolate sensory neuron from rat’s dorsal root ganglia (DRG) using different concentration of collagenase

1.3.2 Specific Objectives

a. To isolate dorsal root ganglion neurons (DRGs) from mice.

b. To dissociate the isolated cells using different collagenase concentration

c. To observe axonal growth of isolated neuron

d. To compare cell viability between 3 different concentration of collagenase

1.4     HYPOTHESIS

An increase in concentration of collagenase will increase the cell death of dorsal root ganglia (DRG).

CHAPTER 2 : LITERATURE REVIEW

2.1 PERIPHERAL NERVOUS SYSTEM

The nervous system is a complex network of nerves which is essentially the most powerful communication system as it controls and coordinates every parts of the body. The nervous system mainly consists of two components which are central nervous system (CNS) that consists of the brain and spinal cord, and peripheral nervous system (PNS) that consists of the nerves and ganglia located outside of the CNS. Generally, the PNS essentially serving as a relay between the brain and spinal cord and the rest of the body which functions  to connect the CNS to the limbs and organ. It transmits information from the central nervous system (brain and spinal cord) to every other part of the body. Unfortunately, unlike the CNS, PNS is more susceptible to mechanical injuries and toxins as it is not protected by the vertebral column and skull, or by the blood-brain barrier. However, adult mammalian peripheral nerves are capable of regenerating and regaining gradual functional restoration after lesion compared to the central nervous system (Luo, Zhu, Zhang, & Jin, 2015).

2.1.1 Cellular composition in the peripheral nervous system (PNS)

Peripheral nervous system is a part of nervous system which is located outside the brain and spinal cord. The structural composition of the peripheral nervous system includes the cranial nerves, spinal nerves and their roots and branches, peripheral nerves, and neuromuscular junctions. The human brain contains about 100 billion neurons which are the basic working unit of the nervous system.

Neurons are the core components of the nervous system that includes the brains, spinal cord in which together comprise the central nervous and ganglia of the peripheral nervous system. In a neuron, a cell body or soma containing nucleus, and extensions called axons and dendrites which help the neurons to communicate with each other can be found. Dendrites are thin structures that arise from the cell body of a neuron which serve as the receiver or input portions of the neuron. The synaptic knob at the ends of each dendrites are the “connections” to adjoining nerve. Specifically, dendrites receive signals from other neuron to the cell body whereas axons take signals away from the cell body. An axons join the cell body (soma, perikaryon), at a cone-shaped region called axon hillock. Bundles of nerve fibers or axons in the peripheral nervous system play a very important role in conducting information to and from the central nervous system.

Two classes of neurons can be found in human peripheral nervous system includes sensory neurons which detect incoming light, sound, odor, taste, pressure, and bring the signals into the CNS and  the motor neurons which carry the signals out of the CNS to control voluntary movement. Motor neurons which control the skeletal muscle are located in the CNS, while the sensory neurons located outside of the CNS, found in clusters known as ganglia. Ganglia are the bundle of nerve cell processes which derived from collections of nerve cell bodies. A nerve is an organ that compose multiple nerve fibers which forced together into sheaths of connective tissue. Endoneurium is the sheath that houses blood capillaries that feed nutrients and oxygen to the nerve. The fibers are bound into fascicles and covered with perineurium and the entire nerve wrapped in epineurium.

The sensory (afferent) are subdivided into somatic and visceral divisions in which the somatic sensory division function in carrying the signals from receptors in the skin, muscles, bones and joints while the visceral sensory division carries signals from viscera of the thoracic and abdominal cavities. Same goes to the motor (efferent) divisions in which the somatic motor division carries signals to the skeletal muscles while the visceral carries signals to glands, cardiac muscle, and smooth muscle.

2.2 DORSAL ROOT GANGLIA (DRG)

In human, there are 31 pair mixed spinal nerves carrying autonomic, motor, and sensory information between the spinal cord and the periphery, 8 cervical spinal nerve pairs, 12 thoracic pairs, 5 lumbar pairs, 5 sacral pairs, and 1 coccygeal pair. The spinal nerves derived from afferent sensory dorsal axon and motor ventral efferent axons in which the dorsal root ganglia (DRG) is formed when the dorsal sensory root exits the neural foramina.

  Bundles of nerve cell processes developed from collections of nerve cell bodies enveloped in epineurium are collected in the enlargements called ganglia which divided into several categories that are the motor ganglia of the autonomic nervous system, special sensory ganglia of the auditory and general sensory ganglia associated with cranial nerve and spinal nerves which the dorsal root ganglia resides. Dorsal root ganglia (DRG) are located along dorsal roots of the cervical , lumbar, thoracic, and sacral spinal nerves near their entry to the spinal cord whereas the cranial nerve ganglia are located along the roots of cranial nerve. (Papka, 2009).

The spinal ganglia or posterior or dorsal root ganglia (DRG) are the collections of afferent sensory fibers which can divided into three categories including large neurons which involved in proprioception (Hammond, Ackerman, & Holdsworth, 2004), small neurons that contain neuropeptide and another one small neurons that do not contain the neuropeptides (Snider & Mcmahon, 1998). DRG contain the unipolar neurons of sensory fibers which passes through the ganglion without synapsing to carry signals to the cord in contrast with automic nervous system which the preganglionic fiber enters the ganglion and synapses with other neuron.

2.3 PERIPHERAL NERVE INJURIES (PNI)

The peripheral nervous system (PNS) has an intrinsic ability for repair and regeneration. Peripheral nerve injuries correspond to a substantial clinical problem with inadequate or unsatisfactory treatment options. (Hu et al., 2016) stated that peripheral nerve injury mostly caused by direct mechanical trauma and less frequently by congenital defects or secondary tumor resection. In North America, approximately 2-5% of trauma patients experience PNI and about 100,000 surgeries are performed every year (Hu et al., 2016). As stated by (Kaplan, Egles, & Marin, 2014), peripheral nerve injury such as trauma can give rise to distal atrophy of target muscle or loss of sensation which affect 2.8% of trauma USA patients annually. For each year, these peripheral nerve damage affect 300,000 patients in Europe and 200,000 patients in USA (Kaplan et al., 2014).

Peripheral nerve injury is often indicated by loss of sensation, partial or complete apraxia, chronic pain, and occasionally permanent disability which can be caused by several conditions include diabetes, Guillain-Barr’e syndrome and cancer along with iatrogenic injuries (Bhangra, Busuttil, Phillips, & Rahim, 2016). According to (Faroni, Mobasseri, Kingham, & Reid, 2014), potentiality for regeneration associates to the age of patients, mechanism of injury, and in particular to the proximity of the injury to the nerve cell body. Distal digital nerve injuries will result in sensory loss to a fingertip and will regenerate well whereas proximal brachial plexus avulsions are functionally damaging with impaired hand sensation, reduced motor function and recurrent pain and cold intolerance due to the torn of nerve from its attachment at the spinal cord which may have intense and life-long effect on the patient in terms of their ability to perform activities and works.

Concerning the problems, various surgical approaches have been developed in order to repair peripheral nerve injury including end-to-end nerve suture repair, which is the favored option for nerve gaps smaller than 5 mm whereas for gap greater than 5 mm, autologous transplantation considered as the “gold standard” for the treatment. However, several common side effects such as donor site morbidity and neuroma formation in the donor region may presents (Luo et al., 2015). Therefore, on account of these limitations, huge strategies has been devoted to the development of synthetic guidance conduit such as nerve guidance scaffolds, physiochemical and biological cues have been applied for the repair  of peripheral nerve injuries  (Hu et al., 2016).

Tissue engineered conduits have been employed with promising results for bridging peripheral nerve damage by providing physical guiding and biological cues. Mesenchymal et al,.(2006) stated that the goal of peripheral nerve tissue engineering is to create a scaffold that guide the sprouting fibers to their end organ and offers nutritional support and up to present, two concepts have been established which is (1) a scaffold combined with certain growth factors to enhance regeneration and (2) a scaffold with cultured Schwann cells (Mesenchymal et al., 2006).

2.3.1 Dorsal root ganglia (DRG) in tissue engineering

Dorsal root ganglia (DRG) has an important role in the modulation of peripheral and central sensory processing including inflammation, somatic pain, the development of abnormal neuropathic pain. Consequently, DRG has been suggested to be an active and not a passive organ in the process of development of chronic pain denying the thought in the not too distant past which suggest that DRG as a passive organ that do not involved in the development of abnormal aberrant neuropathic pain (Krames, 2014).

DRG is a great clinical target for pain control, delivery of anti-inflammatory steroids, and surgery. The greatest proportion of the body’s sensory neurons which are responsible for the transduction of the information from the periphery to the central nervous system  are compose n the DRG. Unlike the CNS which are protected with the blood-brain barrier, the DRG is not protected with any protective surrounding capsular membrane which may expose the DRG neurons to toxic agents and their special position also exposes them to injury. Changes during development and variety of pathologies and  injuries can seriously affected the excitability of the DRG (Haven, 2009).

Rodent DRG neuron have been used in order to examine the sensory nerve development, function and regeneration, to aid in drug discovery (Manuscript, 2010) and to clarify the mechanisms of uderlying peripheral nerve disorders (Sleigh, Weir, & Schiavo, 2016). Isolated of sensory neuronal preparations had been increased in order to understand examine the cellular mechanisms involved in pain signaling as these in viro preparations have numerous advantages in which it can determine whether various inflammatory mediators and algogenic agents have direct actions on sensory neurons. With in vivo preparations, it is hard to interpret whether the specific compound acts directly through generating other agents because several compounds may act on multiple cellular targets in tissue preparations and often generate other inflammatory mediators. Besides, the intracellular signaling pathway for agents that modulate the excitability of sensory neurons can be examined. Other than that, the concentration of mediators and drugs that are used to alter the cell function can be well controlled without having to deal with pharmacokinetics variables that occur in vivo or during administration of drugs in isolated tissue preparation (Burkey et al., n.d.).

2.4 COLLAGENASE

2.4.3 History of collagenase

Collagenase are endopeptidase which digest and cleaves the native collagen  in the triple helix region. Collagens are the major fibrous component of extracellular connective tissue in animal. The collagenases of the bacterium Clostridum histolyticum  are of popular interest besides the mammalian and amphibian tissue collagenases and have been subject of studies for more than 40 years. The characteristics of clostridial collagenase which is presently known today as the most powerful collagenase were comes from the pioneering studies in the 1950s by Mandl, Seifter, Harper and their partners and the commercially available collagenase isolated from anaerobic bacterium Clostridium histolyticum was first proposed by Worthington in 1959. In the mid 1980s, Grant, Auburn, Yoshida and their associates found that several separable collagenases exist and these fractions’ specificities and stabilities were partially characterized. According to Bon and Van Wart, the collagenase were classified as either Class I or II based on the varies properties which differ in aspect of their activities, stabilities, and amino acid composition, but also share numerous similarities. Clostridial collagenase has gained most interest in 1962 when Gross and Lapiere obtained evidence for the discovery of the first vertebrate collagenase in bullfrog tadpole tissue culture media. In addition, collagenase from bacteria is unique rather than other collagenase because it can degrade both water-soluble denatured ones and water-insoluble native collagens and also able to attack almost all types of collagens besides capable of breaking multiple cleavage within the triple-helicle. Consequently, collagenase is widely used in isolation of tissue from animal.In 1970s, numerous collagenases were then found in other bacteria, marine life, amphibians and mammals by Schoellmann, Fisher, Welton and Woods following the discovery by Gross and Harper. Further studies of collagenases from human and other mammalian sources were continue to be actively studied to get better understanding of the pathology and treatment of human disease, for example the relationship between collagenase and rheumatoid arthritis (Possible, 1973), metastasis, wound debriding, herniated disc treatment, angiogenesis, tissue repair, and cirrhosis.

2.4.4 Applications of collagenase in tissue engineering

In tissue culture research and cell biology studies, tissue dissociation or primary cell isolation and cell harvesting are the principal applications for enzymes which is best achieved by means of the tissue-dissociating collagenases, assisted by other proteolytic enzymes. The goal of cell isolation procedure is to maximize the yield of functionally viable, dissociated cells. On account of that, the choice of one technique over another and modifications that could lead to even better results is very important to be understood in order to yield maximum results (Oseni, Butler, & Seifalian, 2013). One of the primary parameter contributing to the success of the present study was the concentration of the enzyme used (Yonenaga et al., 2010). Collagenase is used to mediate tissue dissociation, which is one of the crucial steps in cell isolation procedures that will affect the cells yield, viability and functions. In addition, viability and function of the cells is very important in order to use in research studies, development of new drugs, transplantation and tissue engineering.

In tissue engineering, distinctive cell types can be isolated from tissues or organs from different human or animal tissues cultured under biochemical and biophysical stimulation that will ultimately producing functionally identical and comparable tissues or organs. Collagenase capable to cleave and breaking down the triple helical bond in collagen molecule which has been successfully used for bone cells isolation, cartilage, thyroid glands (Kerkof, 1982), ovarian and uterine tissues (Woods & Tilly, 2013), skin, endothelial cells, neuronal cells, and others.

Every tissue have collagen which is the most abundant protein of vertebrates containing collagen fibrils as the main components of the supporting tissue of connective tissue, bones, cartilage, teeth and extracellular matrices of skin and blood vessels. These collagen fibrils are complex structure surrounded by extrafibrillar matrix that contain proteoglycan which may interact specifically with hyaluronic acid to form aggregates. Unfortunately, the complex composition of the connective tissue have to be dissociated in research or clinical situations. Consequently, the tissue need to be incubated with collagenase in order to weaken and softened the collagen structure of the tissue while permitted gentle dissociation (Centre, 1977).

CHAPTER 3 : METHODOLOGY

3.1 STUDY DESIGN

The experiment required dorsal root ganglia (DRG) located in the dorsal intervertebral foramen adjacent to the spinal cord. Sprague Dawley rats will be sacrificed using asphyxiation technique to harvest the DRG at the spinal column. The rats will not be given any chemicals and fed normally. The DRG obtained will be subjected to enzymatic tissue dissociation using three different concentrations of collagenase. Isolated cells will be then cultured in Dulbecco Modified Eagle Medium with D-valine. DRG neuron will be observed and further characterized through immunochemistry analysis. Lastly, statistical analysis and quantitative measurement of axonal will be carried out.

3.2 LIST OF REAGENTS, MATERIALS AND APPARATUS

3.2.1 Reagents

Dulbecco Modified Eagle Medium (DMEM), D-valine, Penicillin-streptomycin, Amphotericin B, Fetal bovine serum (FBS), Phosphate buffer saline (PBS), Laminin, Poly-L-lysine, collagenase

3.2.2 Materials and apparatus

Laminar flow hood, centrifuge machine, stereomicroscope, desiccator, inverted microscope, cell incubator with 37°C and 5% CO2, water bath, and dissecting sets.

Pipettes, micropipettes, sterile tissue culture 6-well plate, sterile petri dish 100mm diameter, pipette tips, centrifuge tubes, and gloves.

3.3 REAGENT PREPARATION

3.3.1 Collagenase

A total of 10 ml of collagenase with concentration of 0.025%, 0.05% and 0.1% (w/v) will be produced by adding 2.5 mg, 5 mg and 10 mg of collagenase powder to 10 ml of pure DMEM. The mixed solution will be sterilized by using 0.2 μm syringe filters. The collagenase is prepared a day before cell isolation.

3.3.2 Poly-L-lysine

Poly-L-lysine can be used directly without prior preparation. it will be aliquoted in 1 ml and will be stored at 4°C.

3.3.3 Penicillin-Streptomycin

Penicillin-Streptomycin will be aliquoted into sterile Eppendorf tube making 1 ml aliquots and stored at -18°C. 5 ml of this solution will be used in making 500 ml of DMEM. Penicillin-Streptomycin served as an antibiotics which will prevent bacterial contamination to the culture.  

3.3.4 L-glutamine

100 ml of 200 mM L-glutamine will be aliquoted into sterile Eppendorf tube making 1 ml of aliquots. Then it will be stored in the freezer at -18°C. 1 ml of this solution will be used in making 500 ml of DMEM to be served as an amino acid supplement in culturing cell

3.3.5 Cell culture medium

Dorsal root ganglia (DRG) culture medium will be prepared with contents of 1% Penicillin-Streptomycin, 1% Amphotericin B, 1% L-glutamine, 10% Fetal bovine serum (FBS), and D-valine. This sterile medium will be stored in 50 ml schott bottle at 4°C for a maximum of one week.

3.3.6 Culture coating

The coverslip will be pre-coated with poly-L-lysine and incubated for 30 minutes to be added into a 6 well plate. Coating preparation will be done one day before cell seeding. Then, the coverslip will be washed with sterile water and allowed to dry before coated with 50 μl of Laminin and incubated for another 30 minutes to be added into 6 well plate. Laminin solution will be added on the day of cell seeding.

3.4 FABRICATION OF COLLAGEN SCAFFOLD

3.4.1 Extraction of collagen

The tail of a rat will be dissected by making a full-length incision through the skin and will be stripped away to remove and discard the skin exposing the tendons with the white fibres (collagen fibres). Then the collagen fibres will be cut into pieces and collected into a beaker containing PBS.  Next, the dissected tendons will be then placed in 500 ml of 0.5M acetic acid for a couple of days with stirring using magnetic stirrer to dissolve the tendons. The solution will be filtered through cheesecloth to remove any insoluble material before poured into dialysis tubing to be dialyzed against 1 litre of Dialysis Buffer (Sodium Phosphate Dibasic (Na2HPO4) and 11.5 mM Sodium Phosphate Monobasic (NaH2PO4) adjusted to pH 7.2) to made collagen type I solution.

3.4.2 Dialysis of Collagen Type I

The collagen will be dissolved in 500 ml of 0.15M Acetic acid overnight before adding 25 g of Sodium Chloride (NaCl) to allow the collagen to precipitate and centrifuged at 4000 revolutions per minute for 25 minutes. All supernatant will be removed to leave only the pellet at the bottom of the tube to be collected. The process will be done two times repeatedly. The collagen solution will be the poured into dialysis tube to be dialyzed against 1 litre of Dialysis Buffer for 5 consecutive days to remove all traces of Sodium Chloride (NaCl)

3.4.3 Strerilization of Collagen

After 5 days of dialysis process, the collagen solution will be collected to be centrifuged at 4000 rpm for 25 minutes. All the supernatants will be removed and the cell pellet is collected to cover with 70% Ethanol in order to sterilized the collagen. Then the collagen will be mixed for 48 hours. After that, the collagen will be centrifuged again at 4000 rpm for another 25 minutes and the collected pellet will be transferred under the laminar flow hood. Lastly, the pellet will be lyophilized overnight and stored for further use.

3.4.4 Neutralization and Gellification of collagen scaffold

5 gram of the collagen extract will be dissolved into 10 ml of 1% of Acetic acid. After that, 9 ml of the collagen mixture will be added with 1.3 ml of 10x PBS. The mixture will be then neutralized with 350 μl of 4M Sodium Hydroxide (NaOH) before incubate at 37°C for 10 minutes.

3.4 COLLECTION OF RAT DORSAL ROOT GANGLIA (DRG)

Adult Sprague Dawley rats will be sacrificed by induction of carbon dioxide via inhalation. The spinal column and the dorsal part will be removed to expose the spinal cord. Then the spinal cord will be divided in half In the saggital plane and the cord tissue will be removed to expose the DRG and roots within the vertebral canals. The individual roots with DRG attached from the vertebral canal will be gently pulled and the DRG from the rat will be collected into culture dish. Next, the nerve roots will be removed by using a dissecting microscope only to leave each DRG intact before transfer to culture dish containing DMEM.

3.5 TISSUE DISSOCIATION

Three different concentrations of collagenase which is 0.025%, 0.05% and 0.1% (w/v) will be added in 10 ml of pure DMEM with D-valine to be incubated with the tissue in a centrifuge tube for 60 minutes at 37°C, and 5% CO2 with agitation. The incomplete digested tissue will be mechanically dissociated by shaking the tube vigorously for every 10 minutes during the incubation period. The cell suspension will be then filtered by using a cell strainer to remove the residual debris before proceeded to be centrifuged at 4000 revolutions per minute for 5 minutes at room temperature forming three separation layer. All supernatants and unwanted materials will be gently aspirated to leave only the neuron cell pellet at the bottom of the tube. Then the cell pellet will be suspended in cell culture media by using micropipette and dispense 2 ml from the suspension into one well of 6-well plate. In order to avoid disruption of the initial cell adherence by the force generated by movement of culture medium, the culture must not be disturbed at this point until day 7.

3.6 CHANGING CULTURE MEDIA

For every two days,1 ml of media will be removed from each well and replaced 1 ml of fresh culture media until day 20 when the cell have reached confluence. After that, estimation of cell count and viability will be made.  which 20 μl of the cell suspension will be resuspended with 100 μl of Trypan blue for viable cell counting by using hemocytometer. The stain will enter the cytoplasm and the cells will take up the stain in dead or damaged cells while live or viable cells do not take up the stain.

3.7 DATA COLLECTION AND ANALYSIS

Data of cells viability is obtained by using MTT assay and observation under fluorescence microscopy. Lastly, statistical analysis will be done using SPSS to determine association in axonal growth between 3 different collagenase concentration.

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