CC BY
16
УДК 57.085.4
Ёнбом Сон1, Л. В. Григорьева2, Чончхоль Дэвид Юн3, Ынчи Цой4, Ёнхва Сон5, Р. А. Марков2,
С. Е. Григорьев2, Ву Сок Хванг4
Подходы к изоляции мезенхимальных стволовых клеток из мышц древней лошади для предупреждения грибковой контаминации
'Институт ветеринарной медицины Национального университета Кёнсан, Корея
2СВФУ им. М.К. Аммосова, г. Якутск, Россия
'Национальный университет Чунбук, Южная Корея
4Фонд биотехнологических исследований Sooam, Южная Корея
5Институт Пастера, Южная Корея
Аннотация. Останки древних животных с мягкими тканями хранят в себе ценную информацию об эволюции и вымирании этих видов. Одним из наиболее уникальных находок ископаемых останков животных плейстоценового периода является полная туша новорожденного жеребенка ленской лошади, вытаявшая в июне 2018 г. в Батагайской котловине Верхоянского района Якутии. По сохранности мягких тканей эта находка, по общему мнению участвующих в исследовании специалистов, является лучшей за всю историю палеонтологии. Сохранились не только все внутренние органы, но и жидкая кровь и продукты метаболизма в жидком нативном состоянии. Нами была проведена изоляция стволовых клеток для попыток клонирования этого вымершего вида лошади. Мезенхимальные стволовые клетки (МСК) являются потенциальными донорами клеток для терапевтического клонирования (переноса ядра соматической клетки) и геномного протеомного анализа. Однако многие тысячелетия нахождения туши
ЁНБОМ Сон - исследователь отдела териогенеалогии и биотехнологии Института ветеринарной медицины Национального университета Кёнсан.
E-mail: ybson@gnu.ac.kr
YOUNG-BUM Son - PhD, Doctor of Veterinary Medicine, Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea.
ГРИГОРЬЕВА Лена Валерьевна - к. м. н., в. н. с. МЦКП «Молекулярная палеонтология» Научно-исследовательского института прикладной экологии Севера СВФУ им. М.К. Аммосова.
E-mail: lenagrigor@bk.ru
GRIGOREVA Lena Valerievna - Candidate of Medical Sciences, Chief Research Scientist, Center of Molecular Paleontology, Institute of Applied Ecology of the North, M.K. Ammosov North-Eastern Federal University.
ЮН Чончхоль Дэвид - PhD, исследователь Института стволовых клеток и регенеративной медицины Национального университета Чунбук, Южная Корея.
E-mail: jdyoon86@gmail.com
YOON Junchul David - PhD, Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, 28644, Republic of Korea.
ЦОЙ Ынчи - исследователь фонда биотехнологических исследований Sooam, Южная Корея.
E-mail: choi88@sooam.com
CHOIEun-Ji - Researcher, Sooam Biotech Research Foundation, 64 Kyunginro, Guro-gu, Seoul 08359, Korea.
древней лошади в условиях многолетней мерзлоты привели к грибковой контаминации тканей, из-за которой попытки получить первичную культуру клеток лошади были неудачными. Была установлена стратегия для предотвращения грибковой контаминации при изоляции МСК. Обработка разными концентрациями амфотерицина Б показала, что при концентрации 2,5 мг/ мл амфотерицин Б проявляет наибольшую эффективность для предотвращения грибковой контаминации. Также был проведен анализ контаминаций в условиях тканевой культуры и условиях культуры из отдельных клеток. Эта оценка доказала, что система культивирования отдельных клеток в значительной степени была защищена от грибковой контаминации изолированных МСК. Результат предполагает его практическое использование для первичной культуры древних животных in vitro для предупреждения грибковой контаминации.
Ключевые слова: древняя лошадь, мезенхимальная стволовая клетка, перенос ядра соматической клетки, долговременное сохранение, мышечная ткань, грибковая контаминация, амфотерицин Б, культура из отдельных клеток, культура прикрепления тканей, система первичной культуры клеток.
DOI 10.25587/SVFU.2019.74.44564
Young-Bum Son1, L. V. Grigoreva2, Junchul David Yoon3, Eun-Ji Choi4, Yeon-Hwa Song5, R. A. Markov2, S. E. Grigoriev2, Woo-Suk Hwang4
Approaches of Mesenchymal Stem Cell Isolation from Ancient Horse Muscle for Preventing Fungi Contamination
'Gyeongsang National University, Jinju, Republic of Korea
2M.K. Ammosov North-Eastern Federal University, Yakutsk, Russia
3Chungbuk National University, Cheongju, 28644, Republic of Korea
4Sooam Biotech Research Foundation, 64 Kyunginro, Guro-gu, Seoul 08359, Korea
5Institut Pasteur Korea, 16 Dawangpangyo-ro 71beon-gil, Seongnam-si, Bundang-gu, Gyeonggi-do
СОН Ёнхва - профессор лаборатории биологии рака Института Пастера, Южная Корея.
E-mail: yhsong@ip-Korea.org
SONG Yeon-Hwa - Cancer Biology Laboratory, Institut Pasteur Korea, 16 Dawangpangyo-ro 71beon-gil, Seongnam-si, Bundang-gu, Gyeonggi-do.
МАРКОВ Роберт Афанасьевич - м. н. с. МЦКП «Молекулярная палеонтология» Научно-исследовательского института прикладной экологии Севера СВФУ им. М.К. Аммосова.
E-mail: ra.markov@s-vfu.ru
MARKOV Robert Afanasievich - Junior Researcher, Center of Molecular Paleontology, Institute of Applied Ecology of the North, M.K. Ammosov North-Eastern Federal University.
ГРИГОРЬЕВ Семен Егорович - к. б. н., г. н. с. лаборатории «Музей мамонта им П.А. Лазарева» Научно-исследовательского института прикладной экологии Севера СВФУ им. М.К. Аммосова.
E-mail: g_semen@mail.ru
GRIGORIEVSemyon Egorovich - Candidate of Biological Sciences, Chief Research Scientist, Lazarev Mammoth Museum, Institute of Applied Ecology of the North, M.K. Ammosov North-Eastern Federal University.
ХВАНГ Ву Сок - генеральный директор Фонда биотехнологических исследований Sooam, Южная Корея.
E-mail: hwangws@sooam.org
HWANG Woo-Suk - CEO of Sooam Biotech Research Foundation, 64 Kyunginro, Guro-gu, Seoul 08359, Korea.
Abstract. From ancient times to modern times, various species of creatures have lived and become extinct. The ancient animals have invaluable information about evolution and extinction of species. Furthermore, Yakutsk is one of the coldest area in the world and Yakutia horses have survived in the cold climate of Siberia and they have evolved to adapt this environment. So there are many of studies about these horses in relation to their external features, metabolic mechanisms and genetic changes. For this reason, we perform stem cells isolation to revive ancient horse, which lived in Yakutia. Mesenchymal stem cells (MSCs) are adult somatic stem cells which exhibit plastic attachment. These cells are potent donor cells for somatic cell nuclear transfer (SCNT) and also have been widely studied for genomic/ proteomic analysis. However, long-term preservation of ancient animal carcass in nature leads to the fungi contamination of tissues, resulting in failure of MSCs primary culture. We established a strategy to prevent fungi contamination of MSCs isolation. The ancient horse muscle tissues were cultured with different concentration of amphotericin b and evaluated for fungi contamination. The treatment of different concentration of amphotericin b showed that 2.5 ug/ml amphotericin b was the most efficient to prevent fungi contamination. Furthermore, we analyze contamination between tissue piece culture condition and single cell culture condition. This evaluation certify that the single cell culture system largely preserves the anti-fungi contamination of MSCs isolation. These results suggest a practical strategy for establishment in vitro ancient animal primary culture system along with prevention of fungi contamination.
Keywords: ancient horse, mesenchymal stem cell, somatic cell nuclear transfer, long-term preservation, muscle tissue, fungi contamination, amphotericin b, single cell culture, tissue attachment culture, primary culture system.
Introduction
Over the last decade, research on various ancient species, including mammoths has been actively conducted [1-6]. Ancient animals have significant information about evolution and extinction of species [7]. Although, fundamental studies about gene and proteins analyses have been conducted to lead understanding about animal's characteristics, the research is not sufficient [8]. One of the main reason for the limitation is that study was performed only using carcass of ancient animals. Therefore, the cloning of ancient animals is focused for the restoration of them. Recently, the investigation of biological activities of nuclei using means of SCNT method is in progress [7].
Yakutia is one of the coldest area in the Siberia and average winter temperatures range +36° to -67,8°. The modern Yakutian horse live in the coldest region and the most resistant to cold [8-10]. Furthermore, modern horse exhibit short limbs with hairy to adapt climate and also show gradual changes in metabolism as the season changes [8-12]. Therefore, many studies have been conducted to analyze the changes in external features, metabolic mechanism and gene.
Equus lenesis (lena horse), which is genetically distinct from modern Yakutian horse, populated Yakutia between 2,000 and 40,000 years ago [4, 5, 13]. The study of these animals is being discussed mainly in connection with the characteristics of Yakutian horse which is currently of interest. Therefore, the investigation of biological changes from cloning of the ancient horse using SCNT is required. To carry out research on the cloning of Equus lenesis through SCNT, we isolate and culture stem cells from muscle tissues to use as donor cell.
Mesenchymal stem cells (MSCs) are adult somatic and multipotent stem cells [14-19]. Recently, cloning with MSCs has been performed in equine [14, 20]. The equine embryos derived using MSCs for SCNT are showed higher blastocyst rates than the equine embryos derived using fibroblasts for SCNT [14, 20]. However, Equus lenesis carcass has been preserved
in the Siberian Far East for long periods and this is exposed to many kinds of pollution sources [4, 5, 7]. Fungi contamination is repeatedly found in tissue culture and important factor of caution in isolation of MSCs [21-23].
In this study, we optimize of ancient foal MSCs primary culture system to prevent fungi contamination. The amphotericin b has anti-fungi capacity and is also effective in MSCs primary culture to prevent fungi contamination [24, 25]. Therefore, we evaluate the efficiency of high concentration of amphotericin b in culture media as anti-fungi supplementation compared to low concentration of amphotericin b. Furthermore, MSCs primary culture using tissues was performed by two types of tissue piece culture condition and single cell culture condition [26-28]. We confirm that the single cell culture system was more efficient to prevent fungi contamination.
Material and methods
Chemicals and media
All chemicals were purchased from Sigma (St. Louis, MP, USA) and media from Gibco (Invitrogen, Burlington, ON, Canada), unless otherwise specified.
Ancient foal
The complete frozen carcass of ancient foal was found in Batagaika megaslump (Verkhoyansky region of Northern Yakutia) at 2018 and now it kept in Mammoth museum of North-Eastern Federal University in Yakutsk (specimen number MM-F23). The foal was approximately two weeks old and it is 98 cm high at the shoulder. Its muscle, all intestinal organs and some hair were preserved. The carcass was preserved at -18° to prevent damage to tissues and organs. After that, the ancient foal was used for collecting soft tissues to establish MSCs.
Isolation of ancient horse muscle derived MSCs
The ancient horse muscle tissues collected from single donor. The isolation of MSCs from all ancient horse muscle tissues was performed following previously described protocols [19, 22, 28-31]. Briefly, tissues were washed three times with Dulbecco's phosphate buffer saline (DPBS) containing 1% antibiotic/antimycotic. The samples were chopped into small tissue pieces and digested in DPBS containing 1 mg/ml collagenase type IV at 37° in an incubator with frequent pipetting at every 15 min interval for 40 minutes. After digestion, the cell suspensions were filtered with/without 40 um nylon cell strainers (BD Falcon, MA, USA) respectively, and further digestion was prevented by adding high glucose Dulbecco's modified eagle's media (high glucose DMEM) containing 10% fetal bovine serum (FBS). The cell pellet was washed 2 times with high glucose DMEM containing 10% FBS by centrifugation at 500 x g for 5 minutes. The each suspended cells were then cultured in high glucose DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 0.25 ug/ml or 2.5ug/ml amphotericin B at 38°, in a humidified incubator at 5% CO2 in air. The cell pellet was cultured for 21 days. After that the medium was changed every 72 hours during the culture.
Fungi contamination
The Fungi contamination was detected by macroscopic and microscopic methods [32]. In brief, the fungi colonies, which located on the media surface and media's turbidity was observed every 24 hours daily with the naked eye. Furthermore, culture vessel was inspected by phase contrast microscope. All samples were evaluated at 7 days interval for 21 days.
Results
Ancient horse tissue
In order to preserve the original shape and survival rate of MSCs, ancient horse cryopreservation was conducted at - 80°. There were no abnormalities in the carcass of the ancient horse. And the skin, muscle, organs and hair were observed in the carcass of the cryopreserved ancient horse (Figure 1). The muscle tissues were successfully isolated from various ancient horse muscle for further studies. The morphology of muscle tissues derived
Fig. 1. Ancient horse and muscle tissues. (A) Cryopreserved ancient horse carcass. (B) Muscle tissue isolated from ancient horse muscle
from cryopreserved ancient horse was analyzed to determine whether the shapes of fresh muscle tissue were similar to those of the muscle tissues derived from cryopreserved ancient horse. There was no ice crystal formation in the tissues and no damage was observed in the muscle tissues (Figure 1).
Effect on amphotericin b supplementation on MSCs primary culture
To evaluate the effect of amphotericin b supplementation on the capacity of MSCs contamination, freshly isolated cell pellet derived from ancient horse muscle were cultured in high glucose DMEM with 0.25 ug/ml or 2.5 ug/ml amphotericin b supplementation for 14 days without medium change. In all cell pellet, after 14 days of culturing, the medium was changed every 72 hours. After the initial plating, the fungi contamination was evaluated at 7 days intervals for 21 days. All cell pellet in high glucose DMEM with 0.25 ug/ml amphotericin b increased the expression of fungi contamination when compared to cell pellet in high glucose DMEM with 2.5 ug/ml amphotericin b in all time-dependent groups (Figure 2). Further, non-contamination was higher in the presence of 2.5 ug/ml amphotericin b (Figure 2).
Fig. 2. Evaluation of fungi contamination. (A) The cultured cell was evaluated by phase contrast microscope. (x50) non-contaminated cells (left), fungi contaminated cells (right) (B) Expression of fungi contamination cell line number in 0.25 ug/ml amphotericin b and 2.5 ug/ml amphotericin b.
Fig. 3. Small tissue attachment culture conditions and single cell culture conditions.
(A) amount of non-filtrated cell pellet (left) and filtrated cell pellet (right). (B) Non-filtrated cell pellet (left) and filtrated cell pellet (right) on the culture dish
Evaluation of contamination of MSCs primary culture with tissue filtration
Our results showed that there was some fungi contamination in cell pellet cultured in high glucose DMEM with 2.5 ug/ml amphotericin b (Figure 2). Therefore, we evaluated fungi contamination to determine between tissue piece culture condition and single cell culture conditions. The suspended cell pellet was filtered with/without 40 um nylon mesh (with/without filtration). The non-filtrated cell pellet revealed dark color compared to filtrated cell pellet and the amount of cell pellet was reduced (Figure 3). The cell pellet was cultured in high glucose DMEM with 2.5 ug/ml amphotericin b and the non-filtrated cell showed increased amount of precipitates when compared to filtrated cell (Figure 3).
We further evaluated fungi contamination in cultured cell at 7 days intervals for 21 days. During the initial 7 days of culture, two fungi contaminated cells were observed in the non-filtrated group. However, there was no fungi contaminated cells in the filtrated group (Figure 4). There was no difference both groups at 7-14 days in contamination number (Figure 4). Whereas, the number of fungi contaminated cells were increased in non-filtrated group compared to filtrated group (Figure 4).
Conclusion
Generally, fungi were important source of caution in primary culture of MSCs. In addition, ancient animal carcass had been cryopreserved in natural environments for long time and they were exposed to pollution sources compared to general animal tissues so careful attention and approach were needed in the tissue primary culture process.
Fig. 4. Evaluation of fungi contamination. (A) The cultured non-filtrated cell pellet (left) and filtrated cell pellet (right) were evaluated by phase contrast microscope. (x50) (B) Expression of fungi contamination cell line number with/without filtration
Conclusively, the present study demonstrated that fungi contamination was decreased when amphotericin b was added at high concentration during primary culture of MSCs. Moreover, filtered single cells using with 40 um nylon mesh reduced fungi contamination compared to non-filtered cell culture conditions. This is the first study in which we have established the primary culture system of ancient horse tissue to prevent mold contamination.
These results suggested that single cell culture system with high concentration of amphotericin b was effective to prohibit the fungi contamination in MSCs isolation derived from ancient animal tissues.
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