मानव सेल संस्कृति घूर्णन सेल संस्कृति सिस्टम: एक मॉडल के रूप में मानव ट्रोफोब्लास्ट कक्ष
Summary
पारंपरिक, दो आयामी सेल संस्कृति तकनीक अक्सर भेदभाव मार्करों, साइटोकिन्स और वृद्धि कारकों के लिए सम्मान के साथ बदल विशेषताओं में परिणाम. तीन आयामी घूर्णन सेल संस्कृति प्रणाली में सेल संस्कृति (आरसीसी) इन कारकों में से कई की अभिव्यक्ति reestablishes के रूप में एक extravillous ट्रोफोब्लास्ट सेल लाइन के साथ यहाँ दिखाया.
Abstract
The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.
Protocol
Discussion
यहाँ प्रस्तुत संस्कृति तकनीक अत्यधिक आक्रामक EVT की तरह कोशिकाओं के साथ जांचकर्ताओं प्रदान करता है. अब यह मान्यता प्राप्त किया गया है कि भेदभाव का एक नुकसान रासायनिक और आणविक cues के लिए सेलुलर प्रतिक्रि?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम अमेरिकी स्वास्थ्य अनुदान NIH / NICHD # HD051998 (सीएएम के लिए) के राष्ट्रीय संस्थानों द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Cytodex microcarrier beads | Sigma-Aldrich | C3275 | |
| Rotating Cell Culture System (RCCS) | Synthecon | RCCS-D | Includes rotor base, power supply, 4 disposable RCCS units |
| RCCS Disposable Units | Synthecon | Contact Synthecon | |
| 3ml Luer-Lock tip syringe | BD | 309585 | |
| 10ml wide-tip serological pipette | BD | 357504 | |
| MEM Alpha | Invitrogen | 12561-072 | |
| Leibovitz’s L-15 medium, powder | Invitrogen | 41300-039 | |
| H2O, Endotoxin free | Fisher | MT-25-055-CM | |
| Sodium Bicarbonate | Sigma-Aldrich | S-7795 | |
| Peptone | Fisher Scientific | BP1420-100 | |
| Fructose | Sigma-Aldrich | F3510-100 | |
| Galactose | Sigma-Aldrich | G5388-100 | |
| Glucose | Sigma-Aldrich | G7528-250 | |
| HEPES | Invitrogen | 15630-080 | |
| L-Glutamine | Invitrogen | 25030 | |
| Insulin-Transferrin-Sodium Selenite (ITS) | Sigma-Aldrich | I1884 | |
| FBS | Invitrogen | 10437 | |
| Penicillin-Streptomycin | Invitrogen | 15140 |
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