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< Title Page for School of Life Sciences>
INTERNSHIP REPORT
IN VITRO STUDY OF MARCHANTIA PALEACEA POTENTIAL AS CYTOPROTECTIVE AGENTS ON HACAT
CELL LINES UNDER OXIDATIVE STRESS
By Sun Joshua
19010135
Submitted to
i3L – Indonesia International Institute for Life Sciences School of Life Sciences
in partial fulfillment of the enrichment program for the Bachelor of Science in
Biotechnology
apt. Pietradewi Hartrianti, S.Farm., M.Farm., Ph.D.
Mario Donald Bani, S.P., M.Biotech. (Adv.)
Internship Project Supervisor: Mario Donald Bani, S.P., M.Biotech. (Adv.) Internship Project Field Supervisor: apt. Pietradewi Hartrianti, S.Farm., M.Farm., Ph.D.
Sun Joshua
Jakarta, Indonesia
INSTITUT BIO SCIENTIA INTERNASIONAL INDONESIA Jl. Pulomas Barat Kav. 88 Jakarta Timur 13210 Indonesia
+6221 295 67888, +6221 295 67899, +6221 296 17296 www.i3l.ac.id
Certificate of Approval
Student : Sun Joshua
Cohort : 2019
Title of final thesis project : Efek Anti Penuaan Bryophyta Anti Aging Effects of Bryophytes
We hereby declare that this final thesis project is from student’s own work. The final project/thesis has been read and presented to i3L’s Examination Committee. The final project/thesis has been found to be satisfactory and accepted as part of the requirements needed to obtain an i3L bachelor’s degree.
Names and signature of examination committee members present:
1 Thesis Supervisor : Mario D.B. , S.P., M.Biotech(Adv) Approved
2 Field Supervisor : apt. Pietradewi H. M.Farm., Ph.D. Approved
3 Lead Assessor : Elizabeth S. B.Sc., M.Sc. Approved
4 Assessor 2 : Richard S. S.TP., Ph.D. Approved
Acknowledged by, Head of Study Program, Ihsan Tria Pramanda, S.Si., M.Sc.
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COPYRIGHT NOTICE
A copy of this final report has been supplied on the condition that anyone who consults it, is understood to recognize that the copyright of this thesis rests with the author.
No quotations either from its hardbound or soft copy should be published without the author’s permission and any information derived from it should be acknowledged and cited properly. Inquiries concerning the usage of this thesis should be addressed to the author at [email protected].
© 2022
Sun Joshua
All rights reserved
STATEMENT OF ORIGINALITY
Submitted to
Indonesia International Institute for Life Sciences (131)
1, Sun Joshua, do herewith declare that the material contained in my final report manuscript entitled:
IN VITRO STUDY OF MARCHANTIA PALEACEA POTENTIAL AS CYTOPROTECTIVE AGENTS ON HACAT CELL LINES UNDER OXIDATIVE STRESS"
His original wori was performed by me under the guidance and advice of my field supervisor, apt.
Pietradewi Hartrianti, S.Farm., M.Farm, Ph.D. and faculty supervisor, Mario Donald Bani, S.P, M.Biotech.
have read and do understand the definition and information on use of source and citation style published by ial. By signing this statement I unequivocally assert that the aforementioned thesis manuscript conforms to published information.
i3L has my permission to submit an electronic copy of my thesis manuscript to a commercial document screening service with my name included. If you check NO, your name will be removed prior to submission of the document for screening.
ZYes
No
Name of student :Sun Joshua
Student iD :19010135
Study Program :Biotechnology
Siened.
AMERERAT
TEMPELCDAKOCZ3SS41796
Sun Joshua
Date:01-02-2023
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ABSTRACT
In this study, the potential of Marchantia paleacea, a bryophyte that grows in the Cibodas Botanical Garden, was studied for its cytoprotective capability against oxidative stress. H2O2 was used. Although bryophytes have been used in traditional medicine for centuries, this group of plants is still poorly studied. Bryophytes have been shown to contain metabolites such as flavonoids, polyphenols, alkaloids, and saponins that can be used as anti-aging agents due to their anti- inflammatory effects. It was hypothesized that M. paleacea may have anti-aging properties that can be used as a cytoprotective agent against oxidative stress. To test the cytoprotective effect of M.
paleacea, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) assay was used. To induce the oxidative stress, hydrogen peroxide (H2O2) was used in this study. The results show that M. paleacea extracts may have cytoprotective effects against H2O2
at 22 ppm. However, further study and validation was still needed to validate the biological significance of the results.
Keywords: M. paleacea, MTS, cytoprotective, cytotoxic
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ACKNOWLEDGEMENTS
The author of this report would like to express gratitude to Indonesia International Institute for Life Sciences (i3L) and Badan Riset dan Inovasi Nasional (BRIN) for giving the author a chance for making this project as his enrichment program.
The author also would like to give thanks to Miss. apt. Pietradewi Hartrianti, S. Farm., M. Farm., Ph.D. and Mr. Mario Donald Bani, S. P., M. Biotech. (Adv.) for the assistance and guidance during the process of the project from the beginning to the end.
The author also wants to give thanks to all students who are involved in this project, for their
guidance and help during the laboratory activities.
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TABLE OF CONTENTS
COPYRIGHT NOTICE 1
STATEMENT OF ORIGINALITY 2
ABSTRACT 2
ACKNOWLEDGEMENTS 3
TABLE OF CONTENTS 4
LIST OF ABBREVIATIONS 6
I. INTRODUCTION 7
II. INTERNSHIP ACTIVITIES 8
III. PROJECT DESCRIPTION 9
A. Introduction 9
B. Methodology 11
C. Results & Discussion 13
D. Conclusion and Recommendation 16
IV. SELF REFLECTION 17
V. CONCLUSION & RECOMMENDATION 18
REFERENCES 19
APPENDICES 22
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LIST OF FIGURES, TABLES, AND ILLUSTRATIONS
Figure 1. MTS to formazan conversion
Figure 2. M. paleacea cytotoxic test MTS results Figure 3. Graph for the Peroxide Cell Viability results
Figure 4. Linear Regression Model of the Peroxide Cytotoxic test Figure 5. Cell viability for cytoprotective test
Figure 6. HaCaT cells under microscope
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LIST OF ABBREVIATIONS
BRIN (Badan Riset dan Inovasi Nasional)
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)
ROS (Reactive Oxygen Species) MP (Marchantia paleacea) ANOVA (Analysis of Variance)
DMEM (Dulbecco's Modified Eagle Medium) DMSO (Dimethyl Sulfoxide)
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I. INTRODUCTION
Badan Riset dan Inovasi Nasional (BRIN) is a cabinet-level government agency formed in 2019. BRIN was formed to facilitate research from many fields, mainly to increase the value of Indonesia's natural resources in order to increase the national economy. BRIN was directly formed by the current president of Indonesia, Joko Widodo, to help the central government in terms of research and development, assessment and literacy, also realization of new inventions or innovation. BRIN has a vision to make the realization of a National Research and Innovation agency that is reliable, professional, innovative and has integrity in serving the President and Vice President, to realize the President's Vision and Mission: "A developed Indonesia that is sovereign, independent, and has a personality based on mutual cooperation". The vision was accompanied by the mission statements (as quoted from BRIN’s website) to achieve it, which consists of:
1. Providing fast, accurate and responsive technical and administrative support as well as analysis to the President and Vice President in carrying out research, development, assessment and application, as well as inventions and innovations, nuclear operations, and integrated national space operations as well as monitoring control and evaluation on the implementation of BRIDA's (Badan Riset dan Inovasi Daerah) duties and functions.
2. Improving the quality of human resources and infrastructure for research and innovation in implementing nationally integrated nuclear and space affairs and fostering the implementation of BRIDA's duties and functions.
3. Providing effective and efficient services in the areas of supervision, general administration, information, and institutional relations.
As a research institution, BRIN’s main activity has been focused on developing new technology, studying new medication candidates, and also improving research culture in Indonesia. In this project, BRIN has been working with i3L in studying the potential of two bryophytes species from Cibodas Botanical Garden, which consist of Marchantia panacea and Pogonatum neesii. The students involved in this project were tasked with carrying out the research that investigates anti-aging properties of these two species through cytoprotective tests.
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II. INTERNSHIP ACTIVITIES
The experiment was conducte in the i3L cell culture laboratory. The project was in cooperation with BRIN and therefore, given to the student through the lecturer in i3L who also acted as the field supervisor. In this internship, the student did not have any fixed schedule. The student was encouraged to arrange their own timeline based on their deadline. The student’s schedule was based on the time when to maintain the HaCaT cell lines, which is done once every two days.
Usually, the cells will be passaged every Monday, Wednesday, and Friday. On weekends, the cells are let to grow for one more day. When the cells are ready for MTS assay, the cells will be seed onto a 96-well plate and incubated for 24 hours afterwards. The schedule for seeding the cells must coincide with the time to maintain the cells in order to make sure enough cells were present. One MTS assay will need 3 days. This is because the cells need to be incubated for 24 hours after being seeded, and 24 hours incubation again after treating it with the bryophytes extracts and H2O2. On the third day, the MTS reagent will be added to start the test. Usually, the cells will be seeded on Monday or Wednesday to avoid working on weekends. However, if needed, seeding the cells on Friday also could be done. In this internship, the student was tasked to perform a cytoprotective test by using H2O2 and bryophytes extract, given from BRIN. In this internship, the student realized that there are so many things that seem easy to be done theoretically. In reality, so many protocols are much harder to be done, and so much trial and error needed to be done. The student had some difficulties at the beginning of the internship due to the lack of laboratory experience. However, with the help of the research assistant and other students from the same internship, the student can finally conduct experiments in the laboratory and gain valuable experience.
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III. PROJECT DESCRIPTION
A. Introduction
Aging or also called senescent, can be defined as a chronic normal culmination where an organism loses its regenerative and bioprotective mechanisms over time as they grow and develop (Flint & Tadi, 2021). This means, as an organism ages, their physiological function to survive and reproduce will deteriorate over time (Flint & Tadi, 2021). On skin, aging can be characterized by wrinkling and loss of elasticity. UV radiation and oxidative stress by Reactive Oxygen Species (ROS) could cause skin aging (Liguori et al., 2018).
Besides its function as a physical barrier to the surrounding environment, skin is also regarded as a cosmetic feature, especially in the younger generation (Zhang & Duan, 2018).
However, the increase of air pollution and global warming has a direct impact on skin aging (Schikowski & Hüls, 2020). This means, outdoor activities, especially in the urban environment can pose a risk in the form of premature aging of the skin (Schikowski & Hüls, 2020). Therefore, a protective agent or medication is needed in order to protect skin from premature aging, caused by oxidative stress posed by the increase of air pollution.
Bryophytes have been used in traditional medicine for centuries (Dey & Mukherjee, 2015).
However this group of plants is still poorly studied (Dey & Mukherjee, 2015). Previous study has revealed that bryophytes might contain metabolites such as flavonoids, polyphenols, alkaloids, and saponins, that can be used as an anti-aging agent through antiinflammatory effect (Kim et al., 2021). In this study, Marchantia paleacea was chosen as a species of interest. M. paleacea is a bryophyte that is categorized as liverwort and can be found throughout the world, including in Indonesia. In this study, the extract of M. paleacea was obtained from Cibodas Botanical Garden, given by the BRIN (National Research and Innovation Agency). According to BRIN, M. paleacea was chosen due to its abundance in the Cibodas Botanical Garden, also due to the relatively higher yield compared to the other species in there. The taxonomic classification of the M.
paleacea was as follow (NatureServe Explorer, 2018):
Kingdom : Plantae Phylum : Hepatophyta Class : Marchantiopsida Order : Marchantiales
Family : Marchantiaceae
10 Genus : Marchantia
Species : Marchantia paleacea
It is hypothesized that M. paleacea may have anti-aging properties that can be used as a cytoprotective agent against oxidative stress. However, like the other species of bryophytes, M.
paleacea was still poorly studied. Further tests are needed to prove its capability as a cytoprotective agent. In order to test the cytoprotective effect of M. paleacea, an MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used. MTS assay is a colorimetric assay used to assess cytotoxicity of certain chemicals, based on the ability of living cells to utilize the NADH-dependent cellular oxidoreductase enzymes to convert the MTS reagent into a purple colored compound called formazan, which can absorb wavelength between 490 nm (Mosmann, 1983). Therefore, increasing levels of living cells concentration will result in the increasing level of formazan level, consequently increasing the level of absorbance at 490 nm (Mosmann, 1983).
Other methods for similar purposes include, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide) test. However, the MTT test was less accurate. This is because the resulting purple formazan from MTT is less soluble in water and could crystallize, reducing the accuracy of the absorbance readings (Riss et al., 2013). Therefore, with MTT reagents, DMSO (Dimethyl Sulfoxide) was often needed to solubilize the formazan (Riss et al., 2013). The addition of DMSO also can reduce the accuracy of the final results as DMSO has low toxicity to living cells (Chen & Thibeault, 2013). On the other hand, the MTS reagents were much more accurate due to the formulation that is much more optimized from the manufacturing company (Riss et al., 2013).
The resulting formazan from the MTS reaction in living cells was also much more soluble, making the purple color to be more pronounced, also reducing the need to add DMSO. The more pronounced purple color also makes the absorbance results of the MTS reaction results to be read at lower wavelength, which is 490 nm. However, despite its advantages, MTS still suffers from light sensitivity. The MTS reagent is very sensitive to light like its older counterpart MTT.
Even a small amount of light can change the chemical phase of the reagent, affecting the final results. Therefore, working with MTS reagents need to be done in a dark condition, limiting the visibility for its user (Cory et al., 1991).
To induce the oxidative stress, hydrogen peroxide (H2O2) was used in this study. Naturally, H2O2 is produced by the living cells from aerobic metabolic activity (Zenin et al., 2022). H2O2 is
11 commonly known to cause oxidative stress by producing hydroxyl radicals (Mahaseth &
Kuzminov, 2017). Furthermore, H2O2 also can produce superoxide (O −) that are very reactive and can have detrimental effects on cells by reacting to protein, lipid, and other organic compounds in the cells (Mahaseth & Kuzminov, 2017). Radicals produced by the H2O2 were known to inactivate glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, inhibiting the glycolytic activity of the cells (Andrés et al., 2022).
The cell lines used in this study were HaCaT (Human Keratynocytes) cell lines. HaCaT cell lines were chosen as they are often used in the study of skin biology and also, they can mimic the condition of human skin because human skin epidermis consists predominantly of keratinocyte cells (Yousef et al., 2021). HaCaT cell lines are immortalized human keratinocytes, and have been used to study epidermal homeostasis and pathophysiology for a long time (Seo et al., 2012). HaCaT cell lines come from healthy adult human keratinocytes, which are spontaneously transformed into an aneuploidy immortalized cell line (Boukamp et al., 1988).
B. Methodology
Before the experiment can be start, the HaCaT cells need to be grown in the 25 mL T-flask for at least 48 hours, with the incubator at 37OC to mimic the internal temperature of the human body, and at 5% of CO2 concentration to maintain the pH stability of the culture (ThermoFisher, 2022). The cells were then seeded into a 96-wells to be incubated for 24 hours with the same parameter as previously. The cells were then treated with the desired treatment and incubated again for 24 hours. On the third day, the incubated cells can be then treated with the MTS reagent. The cells was calculated every time it is being maintained to determine the cells amount need to be suspended for a final volume of 5 mL in the 25mL T-flask, and also before the seeding process to determine how much cells need to be suspended to create 10,000 cells/wells. The cell counting process was done with glass hemocytometer and trypan blue.
There are two MTS assay in total for the whole experiment. The first MTS assay was for cytotoxic test of the M. paleacea extracts and H2O2 while the second one was for the cytoprotective test. The cytotoxicity of M. paleacea extracts need to be tested in order to make sure the extracts was not toxic to the cells, while H2O2 test was to determine the LC50 (Lethal Concentration 50%). The MTS reagent will be applied inside the Biosafety Cabinets without turning the light on as MTS reagents are very sensitive to light (Mazloum-Ardakani et al., 2019).
Image from Cory et al., 1991 showed the conversion of MTS reagent into formazan that absorbs
light at 490 nm.
12 Figure 1. MTS conversion into purple-colored formazan in the living cells
The concentration of M. paleacea extract applied to the cells was 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm respectively. The concentration was chosen based on the previous study. The presence of cytoprotective effect was indicated by the increasing level of absorbance at 490 nm as the level of the M. paleacea concentration increased, in the presence of H2O2. Viable cells will metabolize the MTS reagent into purple colored formazan with the help of NAD(P)H-dependent cellular oxidoreductase enzymes (Cory et al., 1991). The resulting formazan will absorb light at 490 nm (Cory et al., 1991). All the treatment process was done inside the Biosafety Cabinet to prevent contamination to the cell culture.
However, before the treatment, cytotoxic data for H2O2 and M. paleacea extract need to be obtained first with MTT assay. The H2O2 was tested for its LC50 (Lethal Concentration 50%) level, while M. paleacea was tested to make sure no toxic effect was present. In this case, LC50 was the minimum concentration needed to kill 50% of the cell's population (Zhang et al., 2007). All the test and treatment was done in triplicate with the presence of negative control to validate the data obtained. The results was assessed and calculated into cell viability percentage with formula by Singh et al., 2018:
The absorbance or the optical density (OD) value needs to be subtracted first with the absorbance value of the blank. The subtraction was done to eliminate the background value that came from the DMEM (Dulbecco's Modified Eagle Medium) media and leave only the absorbance value of the formazan, which are the results of the cells metabolic activity.
For the M. paleacea cytotoxic test, ANOVA (Analysis of Variance) was used to tell if there was any significant difference between the concentration groups for the cytotoxic test with M.paleacea extracts. For ANOVA to work, null hypothesis and alternative hypothesis need to be established. In this case, the null hypothesis stated that there are no significant differences between each concentration group, while the alternative hypothesis stated that there are
13 significant differences between each group of concentration. For the H2O2 cytotoxic test, linear regression models were used to predict the LC50 (Chang & Flores, 2015). Linear regression models can give the approximation of how much concentration is needed to kill 50% of the cell's population.
C. Results & Discussion
M. paleacea was hypothesized to have cytoprotective effects against peroxide in this experiment. However, the M. paleacea extract needs to be tested first to make sure no toxic effects are present, which can kill the cells later on. According to the results, HaCaT cells with the M. paleacea treatment have viability of 116%, 118%, 112.23%, 117.63%, and 114.63%
respectively (see Figure 1), with the concentration of 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm respectively. The cell viability percentage value that was more than 100% was caused by the differences in the initial cell amount in each well. The cell was calculated at around 10,000 cells/well during the seeding process. However, the cell count was manually calculated with hemocytometer which have an accuracy of around 30% (Stone et al., 2009).
Figure 2. M. paleacea cytotoxic test MTS results
According to the results, it can be seen that there were no significant differences between cell viability across the concentration range. Based on the ANOVA analysis, the P-value was below 0.05 (P>0.05), which implies that the null hypothesis failed to be rejected. Therefore, it can be concluded that the cells can live with more than 80% viability with treatment from the M. paleacea extracts
14 alone. However, validation of the process may be needed in the future to validate the results as the test was only done in triplicate.
After that, the cytotoxic was done to assess the cytotoxicity of H2O2 to determine the LC50.
H2O2 was used to induce the oxidative stress on the HaCaT cells. In order to use the H2O2, a standard and equal concentration need to be determined in order to give accurate measurements when using different amounts of M. panacea extracts concentration. The results of the cytotoxic test with H2O2
showed a percentage of viability of 96.86%, 100%, 67.92%, 14.40%, and 0% at 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm respectively.
Figure 3. Graph for the Peroxide Cell Viability results
Based on the results obtained, the cell viability decreased when the H2O2 concentration increases (see Figure 3). At 100 ppm, the HaCaT cells population was totally decimated. The absorbance values for the 100 ppm were almost at the same level as blank (sample with only DMEM media and no cells). The results in Figure 3 were then converted into a linear regression model. The LC50 were calculated by using the linear regression function (see Figure 4). The LC50 was found to be around 44 ppm. This result was relatively aligned to the previous study which studied the cytoprotective effects of oridonin by using HaCaT (Bae et al., 2013). In the previous study, they also test H2O2 toxicity with different concentration and the results was, 1200 μM or equal to 40.82 ppm of H2O2, was enough to kill roughly 45% to 50% of the HaCaT cell population (Bae et al., 2013).
Therefore, 44 ppm was taken as the H2O2 concentration for the cytoprotective test.
15 Figure 4. Linear Regression Model of the Peroxide Cytotoxic test (Y axis = Cell viability; X axis
= Peroxide concentration)
However, due to the mistakes done in the MTS process, the concentration of the H2O2 used throughout the cytoprotective test, resulted in a diluted form with concentration of 22 ppm. Based on the calculation with the linear regression model (see Figure 4), 22 ppm of H2O2 should leave the HaCaT cells with a viability percentage of 74.61%. After the determination of the H2O2 concentration that needed to be used, the cytoprotective test was done to assess the capability of the M. paleacea extracts to give protection against oxidative stress.
Figure 5. Cell viability for cytoprotective test with the M. paleacea (MP) and H2O2 (peroxide) treatment
Based on the results of the cytoprotective test, it can be seen that the cell viability increases from 78.36% at 6.25 ppm, to 93.61% at 100 ppm. It also can be seen that the cells with only H2O2
16 have viability percentage of 77.8%, almost the same as the cells with 6.25 ppm of extracts, which have viability percentage of 78.36%. These results can indicate that at 6.25 ppm of M. paleacea extracts, there was almost no protection at all, while the 100 ppm gave the highest amount of protection. In this result, it is shown that there was a pattern of increasing viability when the concentration of M. paleacea extract was increased. It may have indicated that the M. paleacea did have cytoprotective effects against H2O2 at 22 ppm. The trend patterns shown in this result were relatively similar to other studies that studied cytoprotective effects against H2O2 with HaCaT cells (Bae et al., 2013; Zhang et al., 2020). However, due to the relatively high error bar at 6.25 ppm and 100 ppm, this result also needs to be taken with a grain of salt, and a validation process may be needed to validate the results. The high error bar can be caused by improper pipetting technique, or due to the presence of air bubbles in the sample. The mechanism involving the protection also still needs to be investigated as the extracts used in this study were crude extracts in its diluted form.
Pinpointing the compound that gives the cytoprotective effects may be necessary for future study.
D. Conclusion and recommendation
Based on this study, M. paleacea extracts could potentially give a cytoprotective effect against oxidative stress produced by H2O2. However, there are still errors shown in the results of these studies and therefore, further validation is needed to prove the biological significance of this study. The possible sources of error are improper pipetting technique and the presence of air bubbles in the samples during the absorbance reading process. Furthermore, the extracts used in this study were a crude extract in its diluted form. Future study for investigation for pinpointing the
exact compounds that give the protection may be needed.
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IV. SELF REFLECTION
In this internship, the student had gained knowledge on how to conduct activity in a cell culture laboratory. Two years of pandemic have hindered the student from having experience in terms of laboratory activity. In fact, this internship has helped the student to gain the necessary skills and knowledge for future study that may be related to in vitro study. The schedule was determined based on the time to maintain the cell when it reached confluence. The MTS process was started by seeding the cells into 96-cells. The seeding schedule must coincide with the time to maintain the cell culture in order to make sure there are enough cells, by counting it first with a haemocytometer.
Every MTS assay will need at least three days to be completed. The first day was to seed the cells and incubation for 24 hours, second day for giving the desired treatment and incubation again for another 24 hours, and the third day was for the addition of MTS reagent and absorbance reading.
During the internships, the student has difficulties in conducting laboratory protocols for maintaining the cells and for MTS assay. However, the student manages to learn the laboratory skills through the help of fellow students, research assistant, and also due to the chance given by the field supervisor. Learning from the internet regarding the reasoning behind all the steps used in the laboratory also helps the student to finally fully grasp the skills and experience.
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V. CONCLUSION & RECOMMENDATION
Through this internship, the student had obtained valuable experience. Due to the pandemic, offline laboratory sessions were off limits. In this internship, the student can finally learn how to conduct experiments in the laboratory firsthand. It is concluded that through this internship, the student has obtained the necessary knowledge for his further study, also the necessary experience that is important for laboratory activity. The laboratory activity was conducted with fellow student who are also part of the project, along with the research assistant. Through this project, the student have manage to complete his EP program by obtaining the necessary data through works that are worth 7 CU, and also by doing the necessary biweekly meeting with the faculty supervisor. Meeting with the field supervisor was done every time there were new updates with the project. Meeting with he field supervisor are usually conducted on Monday to update the progress of the project. Overall, this project has contributed a lot for the student to gain the
necessary experience for his further study.
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Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., & Fusenig, N. E.
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Dey, A., & Mukherjee, A. (2015). Therapeutic potential of bryophytes and derived compounds against cancer. Journal Of Acute Disease, 4(3), 236-248.
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Flint, B., & Tadi, P. (2021). Physiology, Aging. Ncbi.nlm.nih.gov. Retrieved 6 September 2022, from https://www.ncbi.nlm.nih.gov/books/NBK556106/.
Kim, S., Hong, M., Kim, T., Lee, K., Park, S., & Hong, S. et al. (2021). Anti-Inflammatory Effect of Liverwort (Marchantia polymorpha L.) and Racomitrium Moss (Racomitrium canescens (Hedw.) Brid.) Growing in Korea. Plants, 10(10), 2075.
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20 Marchantia paleacea a liverwort. NatureServe Explorer 2.0. (2018). Retrieved December 21, 2022,
from
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APPENDICES
Figure 6. HaCaT cells under microscope 100x magnification
