|Year : 2015 | Volume
| Issue : 2 | Page : 61-67
Variation in growth pattern and morphological appearance of primary monolayer cultures of chondrocytes and neural cells isolated from the chick embryo at different stages
Rupendra Shrestha, Asha Palat, Selvam Anbarasan, Solomon F. D Paul
Department of Human Genetics, Sri Ramachandra University, Chennai, India
|Date of Web Publication||5-Jul-2017|
Solomon F. D Paul
Professor and Head of Department of Human Genetics, Sri Ramachandra University, Porur, Chennai-600 116
Source of Support: None, Conflict of Interest: None
Aim: This study was an attempt at investigating the variation in growth and morphology of primary avian chondrocyte and neural cell cultures established under standard laboratory conditions. Methods: For establishing chondrocyte cultures, the tibia were dissected from chick embryos that were 12 (E12), 14 (E14) and 15 (E15) days old. For the neural cell culture initiation, the two lobes of the forebrain were dissected from chick embryos that were 4 (E4), 8 (E8) and 12 (E12) days old. The final characterization of the cells was performed by H&E and CFV staining. Results: In the chondrocyte cultures, two forms of morphology were observed which is reversible in process.The primary variation evaluated in chondrocytes cultures was the effect of the media on the state of the cells. Based on whether the cells were cultured in DMEM or MEM, there was a difference in the transformation of the attached cells from the differentiated to de-differentiated forms. The primary variation examined in neuron cultures was the embryonic age of the tissue and the effect that it had on the proliferation and neurite formation of the cells. E8 embryo neural cells cultures result in well-developed cells with neurite formation along with axonal and dendritic outgrowths with highly complex interconnections between them. Conclusion: Ultimately, this study demonstrated the composition of culture media had an effect on the morphological appearance of the chondrocytes, as well as, confirmed that the culture of primary neurons is best performed using cells from an E8 embryo.
Keywords: Primary monolayer culture, Chondrocytes, Neural cells, cell morphology, Characterization
|How to cite this article:|
Shrestha R, Palat A, Anbarasan S, Paul SF. Variation in growth pattern and morphological appearance of primary monolayer cultures of chondrocytes and neural cells isolated from the chick embryo at different stages. Acta Med Int 2015;2:61-7
|How to cite this URL:|
Shrestha R, Palat A, Anbarasan S, Paul SF. Variation in growth pattern and morphological appearance of primary monolayer cultures of chondrocytes and neural cells isolated from the chick embryo at different stages. Acta Med Int [serial online] 2015 [cited 2020 Jun 1];2:61-7. Available from: http://www.actamedicainternational.com/text.asp?2015/2/2/61/209654
†Authors have contributed equally in experimental work and manuscript preparation
| Introduction|| |
Primary monolayer cell culture, a two-dimensional study of the attachment and proliferation of primary cells, works with cells that have been dispersed mechanically or enzymatically into cell suspension from the original tissue. During the establishment of monolayer cultures, different cell types exhibit variations based on their morphological appearance and growth patterns. These variations are due to different growth requirements which determine whether or not the cells multiply in vitro. The morphology of cells in monolayer cultures depend on various factors such as the source of the cells, age of the tissue from which it was dispersed, substrates and microenvironment etc. Monolayer cell culture has an important role in exploring cellular differentiation and extracellular matrix (ECM) deposition as well as in understanding the molecular mechanism regulating cellular differentiation. However, this system has a disadvantage in that it does not mimic the complex organization found between cells and ECM in in vivo conditions which is entirely dependent on 3D microenvironments.
The culture of chondrocytes and neuronsis a challenging task due to the complexity in their organization. Primary monolayer culture of chick tibia chondrocytes was first described very intelligently by Holtzer and his colleagues in 1960 and laterwas modified by Coon in 1966. Chondrocytes have been observed to exhibit two forms in culture based on morphology - differentiated round cells and de-differentiatedfibroblastic cells.,, These two morphological forms are interchangeable and are influenced by cell density as well as the surface on which the cells are grown.,,
Primary neural cultures derived from chick forebrain neurons were initially demonstrated by Sensenbrenner and colleagues. It has been shown that cultures derived from 8day old chick embryoswere characterized by large, slightly rounded cell bodies with large neurites. The development of the cells begins with the neurons exhibiting lamellipodial and filipodial motility along their marginfollowed by the appearance of short, tapering neurites called ‘minor processes’. Around 50% of the cells progress to a stage where there is extension of a single axon by rapid elongation of one of the minor processes.
This system of two dimensional cell cultures is widely used in the investigation of cell morphology and growth pattern which helps in understanding bone and nerve physiology.
| Methods|| |
Isolation of Primary Cells
12 (E12), 14 (E14) and 15 (E15) day old chick embryos were used. The egg was carefully cracked open over a sterile 100mm petriplate and the embryo was separated from the extra embryonic tissue and placed in a fresh petriplate containing sterile PBS. The embryo was decapitated and epiphyses of tibias of chick embryos which were dissected under sterile conditions.Single cell suspension of chondrocytes was prepared from the tibia by physical mincing [Figure 1]a. The cell suspension containing cell clumps was further treated with 1% trypsin-EDTA and incubated at 37°C for 45 minutes resulting in a uniform cell suspension.
|Figure 1: Single cell suspension after tissue mincing and digestion is shown in a, viability count in b. Morphological features of E12 chondrocytes in DMEM express fibroblastic appearance with few undifferentiated, round cells as shown in c & d|
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4 (E4), 8 (E8) and 12 (E12) day old chick embryos were used. Once the embryo was separated from the extra embryonic tissue and decapitated, the forebrain was dissected out. The two hemispheres of the forebrain form an easily observable triangle of tissue and are separated from the midbrain by a left-to-right fissure. Once the hemispheres were isolated, the PBS was replaced and the thin membranes covering forebrain were dissected off. These membranes are highly vascularized and must be removed in order to reduce the contamination of the future neuron culture with other cell types. Single cell suspension ofneural cells was prepared from the forebrain by gentle physical mincing. The cell suspension containing cell clumps was further treated with 1% trypsin-EDTA and incubated at 37°C for 5 minutes resulting in uniform cell suspension.
Primary Monolayer Culture
The various monolayer cultures were set up in T-25 flasks and six well plates. The six well plates were modified by introducing 2-3 sterile coverslips in each well and coating them with a gelatin solution. After an incubation period of 24 hours, the excess gelatin was rinsed off using sterile PBS. UV treatment of the plates was done for 15 minutes before initiation of the cell cultures. This preparatory step of the six well plates was performed one day prior to the dissection of the chick embryo for both the chondrocyte and neural cell cultures.
Cells were seeded in the density as follows- high density cultures (2.5x 105 cells/cm2), medium density cultures (1.5x 104 cells/cm2) and low density cultures (3 x 103 a cells/cm2). The cultures were set up in DMEM with 10%FBS and were finally incubated at 37° C with 5% CO2 and saturated humidity. The culture media was changed twice or thrice per week. The cultures were observed regularly for signs of contamination or indications that a media change was required. The cultures were monitored daily for signs of contamination (formation of observable particles, change in media colour, microscopic observation) and overall health of the cells in the culture.
Variation - Chondrocytes
The chondrocytes isolated from the E12 chick embryo was cultured in DMEM with 10% FBS, while the chondrocytes from the E14 and E15 chick embryos were cultured in parallel cultures in DMEM and MEM to see the variation in the growth pattern and morphological appearance of the chondrocytes.
Characterization of Growth Pattern and Morphological
The growth pattern and morphological appearance of cells was observed under an inverted phase contrastmicroscope every two days. Microscopic observations were recorded for confluence and morphology both by photography and also by monitoring percentage of confluency.
H & E stain for chondrocytes
Cells grown on coverslips were fixed with 3.7% formaldehyde at 4°C for 40 minutes. The cells were then hydrated in descending grades of alcohol (i.e. 100%, 90%, 80%, and 70%). The coverslipswere rinsed in running tap water and stained with Haematoxylin for about 5-8 minutes. 1% acid-alcohol was used for differentiation and then washed immediately. Bluing was done by treating with Scott's tap water for 2-3 minutes. It was then stained with 1% eosin for 2 minutes and dehydrated in ascending grades of alcohol (i.e. 70%, 80%, 90%, and 100%). Cells were observed immediately and documented.
Cresyl fast violet stain for neuron
The coverslips with attached neural cells were fixed with 3.7% formaldehyde at 4°C for 40 minutes and rinsed with PBS. The coverslips were stained with 0.5% CFV solution for an appropriate time and then rinsed in distilled water. 0.25% acetic acid alcohol was then added for differentiation. The coverslips were then treated with a gradient of alcohol solutions and observed under the microscope. After staining, the morphology of the cells was documented.
| Results|| |
Isolation of Cells and Viability Count
Chondrocytes and Neurons
The total count and cell viability concentration was measured by trypan blue viability assay as illustrated in [Figure 1]b and was found to be 3.5 x 106 cells/ml with a viability of 86% for the chondrocytes. For the neural cells, it was a total cell count of 2x107 cells/ml with a viability of 82%.
Growth and Morphological Pattern of Cells
E12 chick embryo
Fibroblast like morphology was observed within 24 hours of culture in DMEM. The fibroblast morphology retained 80% confluency for upto 5 days with few undifferentiated round cells as illustrated in [Figure 1]c & [Figure 1]d.
E14 chick embryo
Chondrocytes from E14 chick embryo show variation in the growth and morphological pattern between DMEM and MEM. In DMEM, fibroblast like morphology with few undifferentiated round cells was seen in 14 hours of culture and confluency was observed in 3 days as illustrated in [Figure 2]a and [Figure 2]b. However, cultures in MEM show both differentiated round polygonal cells and fibroblast like morphology with few undifferentiated cells. The differentiated polygonal cell morphology was observed by 48 hours and reached a maximum within 3 days. There was a subsequent transformation to a fibroblast like morphology that retains 75% confluency with only a few undifferentiated cells within 7 days illustrated in [Figure 2]c & [Figure 2]d.
|Figure 2: The features of E14 chondrocytes in DMEM and MEM shows variation in morphological appearance and growth pattern. In DMEM, fibroblastic appearance with few undifferentiated, round cells was seen as shown in a & b while in MEM, both differentiated and fibroblastic appearance with few undifferentiated, round cells was observed as shown in c & d|
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E15 chick embryo
Unique morphological and growth patterns were seen in E15 day old chick embryo chondrocytes when compared to E12 and E14 chick embryo chondrocytes. In cultures set up using DMEM the cells initially showed fibroblast like morphology within 24 hours of culture and maintained this morphology upto 48 hours. The cells finally de-differentiated into rounded, polygonal shaped cells and achieved 80% confluency in 9 days as illustrated in [Figure 3]a & [Figure 3]b. In case of cultures using MEM, differentiated, rounded, polygonal morphology was seen in 3 days of culture.Continuous maintenance of its phenotypic morphology was observed until the 9th day along with an 80% confluency as illustrated in [Figure 3]c & [Figure 3]d.
|Figure 3: The features of E15 chondrocytes in DMEM and MEM shows variation in morphological appearance and growth pattern. In DMEM, both differentiated and fibroblastic appearance with few undifferentiated, round cells was seen as shown in a & b while in MEM both differentiated, round and polygonal appearance was observed as shown in c & d|
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E4 chick embryo
The cultures set up were healthy and were maintained for a period of 5 days. The cells were primarily neuroblasts and were initially slow to attach and remained as rounded cells. Attachment was significantly observable from the third day onward along with sprouting of neurites. By the fourth day in vitro,the cells were well grown with neuritic outgrowths connecting the aggregates of cells [Figure a] & [Figure b].
E8 chick embryo
The cells, on plating, remain as unattached, rounded spheres for 1-2 days. Signs of attachment can be observed from the third day onward along with signs of neurite sprouting. By the fourth day,the cells were found to attach individually and form long, thin neurites that connect with nearby cells. Unattached, round cells were found to accumulate over the attached cells in aggregates obstructing them from view. Eventually they attached as well and groups of attached cells with well-defined interconnections were observed from day 6. As the culture progressed, cell attachment and growth increased leading to nearly confluent regions with highly complex interconnections between them [Figure 4]c & [Figure 4]d.
|Figure 4: The morphological features of E4 & E8 neurons in DMEM shows variation in growth pattern. In E4 cells, attachment with sprouting of few neurites was seen as shown in a & b while in E8, neurites with well-defined and highly complex interconnections between them was observed as shown in c & d|
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E12 chick embryo
Once the cultures were set up, two distinct populations could be observed within 24hours - cells that had begun attaching while another type which remained rounded and unattached. After 2days in vitro, increased cell attachment was observed along with the limited formation of neurites between select cells. Neurite formation was not observed on all attached cells. There was a population of cells that were well attached but did not show any signs of developing neurites. By the 4th day of the culture, distinct patches of cells were observed in the culture. These patches were different from the aggregates observed in E8 cultures as they appeared more fibroblast like in nature and were lacking well developed neurites. Observations on the 6th day only confirmed the fact that it was a definite glial population in the culture which proliferated, covering more area in the form of confluent stretches of cells as seen in [Figure 5]a & [Figure 5]b. Neurite outgrowths remained underdeveloped when compared to the cultures from E8 chick embryos.
|Figure 5: The morphological features of E12 neurons in DMEM shows a majority of fibroblast like cells with neurite formation occurring only between a few cells as shown in a & b. Well defined, interconnecting cells with neurites are shown in c & d|
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Occasionally, it has been noticed in a few cultures, the formation of floating aggregates of cells in the form of spheres or neurospheres. These cultures possess very few to no attached cells at all and all the cells seem to be in the form of floating spheres. The spheres are viable and refractile and on disruption by pipetting or enzymatic treatment can be brought into a single cell suspension and seeded into a new culture. In some cases, the spheres seem to have settled down and extended neurites. [Figure 6]a & [Figure 6]b.
|Figure 6: The formation of neurospheres in E12 chick neuron cultures was seen as shown in a & b. The morphology of chondrocytes with clear architecture, nucleus and vacuole after h &e staining was observed as shown in c & d while Cresyl fast violet staining of neuron presents fine nissl granules and nucleus as shown in e & f|
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Characteristics of Cells in Monolayer Cultures
H&E stain for chondrocytes.
The general morphology of chondrocytes was analysed by H&E staining of cells grown on the coverslip. The clear architecture and shape of chondrocytes was observed. A distinct nucleus as well as vacuole was observed as illustrated in the [Figure 6]c & [Figure 6]b.
Cresyl fast violet stain for neurons
Cresyl fast violet staining was performed on the primary neuron culture. This is a stain that binds to the nissl granules of neurons.It also stains the soma of the cell a violet-purple colour and does not stain glial cells. Staining of the culture helped improve the visualization of the structures of the cell as seen in [Figure 6]e & [Figure 6]f.
| Discussion|| |
The establishment of primary monolayer cultures of chondrocytes and neurons under normal conditions in culture media without any supplements is a challenging task. The primary chondrocyte culture was established in two different mediums - DMEM and MEM with 10% FBS. In the present study, two different morphological patterns of chondrocytes were observed- rounded, polygonal and fibroblastic. This study has shown the transformation of rounded, polygonal cells to fibroblastic and reverse. There is no evidence so far that describes which morphology the cells will initially start as, or end. However, in the present study, we found that chondrocytes initially grew as rounded, polygonal cells that transformed to fibroblastic morphology in the case of E14 chondrocytes grown in MEM. The reverse processwas observed in E15 chondrocytes grown in DMEM where the cells transformed fromfibroblastic cells to rounded, polygonal cells. The difference in growth pattern and morphological variation of same cells is seen between two media is might be due to amino acid. As it is known that there is insufficient amino acid and vitamins in MEM as compared to DMEM. Therefore it is further need for the study of amino acid effects on cell morphology. These features of the growth pattern is also altered by the seeding density, type of media used, surface on which cells have attached, environmental influence etc. Similar evidence was shown in a previous study, where the chondrocytes have two forms in culture; ‘differentiated’ with a rounded morphology,, and ‘de-differentiated’ with a stellate or fibroblastic appearance. The two morphological forms are interconvertible and are influenced by cell density and the surface on which the cells are grown,,, oxygen partial pressure, thyroxine, 5-bromodeoxyuridine,, embryo extract,, and other environmental conditions. The differentiated chondrocytes in culture can be grown for a few weeks. The lifetime of differentiated cells can be enhanced by sub-culturing and cloning,, but with progress of time for about a month, there will be only presence of de-differentiated cells. Eventually (nearly always within 1 month) only ‘de-differentiated‛ cells will be present. Several years ago, the in vitro study on the altered behaviour of specialized cells was performed from a different angle with great curiosity.,, A similar study of chondrocytes in vitro depends on the technology available to really understand the establishment of functional cells in vitro.,, To understand whether the reversal mechanism of de-differentiation actually occurs or not, nuclear and cortical transplant studieshave given a clear idea.,
A comparison of the variation in growth and morphology of the primary chondrocytes has been described in [Table 1]. The factors taken into account are the age of the embryo used for establishing the culture and the type of cell culture medium used.
|Table 1: Variation in growth pattern and morphological appearance of chondrocytes|
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The variation in growth and morphology of the primary neurons has been compared and described in [Table 2]. Here, the age of the embryo was the major factor taken into consideration for studying the differences in neurite outgrowth and morphological appearance of the cells.
|Table 2: Variation in growth pattern and morphological appearance of neurons|
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For the primary culture of neuron, the selection of the ideal age of chick embryo needed to optimize the neuron specific monolayer culture was crucial. In the study, different aged embryos such as E4, E8 and E12 were selected to see variation in cell growth rates and difference in morphology. In E4 the cells developed with neuritic outgrowths connecting the aggregates of cells. Cells in this stage were primarily neuroblasts which further divided to grow as specific neuronal cells. These cells were observed to be highly susceptible to environmental conditions. Previous work on E3 embryos provides the evidence of culture from dividing neuroblasts and an observation on their differentiation. The fact that the cells are predominantly neuroblasts that are plastic and susceptible to their environment might have contributed to the early termination of the cultures., In E8 as the culture progressed, cell attachment and growth increased leading to nearly confluent regions with highly complex interconnections between them. In previous study it shows E8 was the optimal stage for isolation of cells that results in a culture of nearly pure neurons. It has been found that while plating at very low densities (1000cells/ cm2) the neurons are incapable of developing axons. The ideal plating density has been found to be around 8-10 x 103 cells/cm2. This cell seeding density is low enough to allow most axons to be clearly associated with a cell body in particular and yet high enough to support axonal development as well. However in E12, neurite outgrowths remained underdeveloped when compared to the cultures from E8 chick embryos and the cultures where observed to have a large population of well grown fibroblastic cells. In the study the typical feature of neurons was observed when the cells were grown in low densities where neurites were easily identified as axons. Similar characteristics of neurite formation from low density cell cultures were shown in a previous study. Theneurospheres were observed in cultures that had just been established or had just undergone a media change. This was an indication that the environmental conditions were less than sub-optimal and the cells preferred to aggregate together in spheres rather than attach to the substrate individually.
The monolayer culture of avian chondrocytes and neurons was established without additional supplements. In the chondrocyte cultures, two forms of morphology were observed which is reversible in process whilein the neural cultures, it was confirmed that E8 was the optimal embryonic stage for establishing cultures.
| Acknowledgements|| |
The authors would like to thank Dr. Alan M. Punnoose, Cell and Tissue Engineering Laboratory, Centre for Regenerative Medicine and Stem Cell Research, Sri Ramachandra University, Chennai, India for his assistance, support and also for providing the media and reagents necessary for the project.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]