Explant culture

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In biology, explant culture is a technique to organotypically culture cells from a piece or pieces of tissue or organ removed from a plant or animal. The term explant can be applied to samples obtained from any part of the organism. The extraction process is extensively sterilized, and the culture can be typically used for two to three weeks.[1]

The major advantage of explant culture is the maintenance of near in vivo environment in the laboratory for a short duration of time. This experimental setup allows investigators to perform experiments and easily visualize the impact of tests.[2]

This ex vivo model requires a highly maintained environment in order to recreate original cellular conditions. The composition of extracellular matrix, for example, must be precisely similar to that of in vivo conditions in order to induce naturally observed behaviors of cells. The growth medium also must be considered, as different solutions may be needed for different experiments.[1][2]

The tissue must be placed and harvested in an aseptic environment such as sterile laminar flow tissue culture hood. The samples are often minced, and the pieces are placed in a cell culture dish containing growth media. Over time, progenitor cells migrate out of the tissue onto the surface of the dish. These primary cells can then be further expanded and transferred into fresh dishes through micropropagation.

Explant culture can also refer to the culturing of the tissue pieces themselves, where cells are left in their surrounding extracellular matrix to more accurately mimic the in vivo environment e.g. cartilage explant culture,[3] or blastocyst implant culture.[4]

Application[edit]

Historically, explant culture has been used in several areas of biological research. Organogenesis and morphogenesis in fetus have been studied with explant cultures. Since the explant culture is grown in the lab, the area or cells of interest can be labeled with fluorescent markers. These transgenic labels can help researchers observe growth of specific cells. For example, neural tissue development and central nervous system regeneration have been studied with organotypic explant culture.[1]

The role of a specific gene, gene expression, and the mechanism of action all can be studied with explant culture as well. Certain factors that control or contribute to growth could be identified during different stages of embryogenesis. Looking at the expression pattern would allow tracking of where the gene transcripts have been. How much gene has been expressed could be quantified too.[1]

Coupling with stem cell research, researchers have successfully grown simple organs derived from autologous human pluripotent stem cells. So far bladder and trachea have been developed. This method attempts to address tissue rejection, and there are already cases of successful transplantation. A research team from Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina, successfully transplanted stem cell-engineered bladders to seven pediatric patients suffering from malfunctioning bladders. Another case was from a team at University College, London, UK, which transplanted a wind pipe derived from the patient’s own stem cells.[5]

Even with all the advantages to explant culture, there still are several caveats. The downside of explant culture is that it does not provide sufficient time to study chronic diseases. Although two to three weeks may be enough time to study acute changes, it is not fit for experiments requiring long-term observations.

General Protocol[edit]

Often, mice are used as model organism to study organ development. It must be sacrificed in order to collect target organ or tissue. After the sample of interest has been gathered, it must be stored at 37 °C incubator with 5% carbon dioxide and a growth medium. Serum-free, nutritious medium provides all the nutrients for the cells to grow. Thus cells would ultimately form a thin sheet. The samples can be also sliced and incubated to observe how thin sheets of cell go through development.[6]

Current Research[edit]

Bone and cartilage[edit]

The explant culture has been used as an ex vivo model to investigate linear bone growth, bone and cartilage metabolism, cancer cells-induced bone disease, stem cell behavior in bone repair, and bone response to mechanical loading. Observing how human bones and cartilage develop over time is extremely difficult, as they require precise set of conditions within the body. Preserving extracellular matrix environment is crucial for three-dimensional organization of these tissues. Understanding how osteoclasts, osteoblasts and osteocytes actively engage together is needed to further study their formation.[7]

Retina[edit]

Many neurobiological processes have been studied with retinal explant cultures. Understanding retina’s development has led the way for researchers to study pathological neurodegeneration and related retinal diseases more closely. Cellular grafts derived from retinal stem cell therapy is an active area of research to treat macular degeneration, retinitis, pigmentosa, and glaucoma.[8]

Heart[edit]

A neonatal mouse ventricle organ explant has been proposed as a model to study gene and cell therapy to address cardiovascular diseases. Although the explant cultures are only good for up to 8 days, they still retain essential physiological functions and structures.[9][10]

References[edit]

  1. ^ a b c d "Explant Culture for Developmental Studies | Protocol". www.jove.com. Retrieved 2017-05-25. 
  2. ^ a b "Explant Culture of Neural Tissue". JoVE Science Education Database. 
  3. ^ Orth MW et al. (2000) Cartilage turnover in embryonic chick tibial explant cultures. Poult Sci 79:990-993.
  4. ^ Ivan Bedzhov, Magdalena Zernicka-Goetz.(2014). Self-Organizing Properties of Mouse Pluripotent Cells Initiate Morphogenesis upon Implantation. Cell,; DOI: 10.1016/j.cell.2014.01.023
  5. ^ Hunter, Philip (2017-05-25). "One organ at a time: Research has been making much progress to create in vitro human tissues for transplantation but laboratory-grown complex organs still remain decades away". EMBO Reports. 15 (3): 227–230. doi:10.1002/embr.201438528. ISSN 1469-221X. PMC 3989688Freely accessible. PMID 24554301. 
  6. ^ "Retinal Explant Culture —BIO-PROTOCOL". www.bio-protocol.org. Retrieved 2017-05-25. 
  7. ^ Marino, Silvia; Staines, Katherine Ann; Brown, Genevieve; Howard-Jones, Rachel Anne; Adamczyk, Magdalena (2016-06-29). "Models of ex vivo explant cultures: applications in bone research". BoneKEy Reports. 5: 818. doi:10.1038/bonekey.2016.49. PMC 4926536Freely accessible. PMID 27408711. 
  8. ^ Garg, Aakriti; Yang, Jin; Lee, Winston; Tsang, Stephen H. (2017-02-02). "Stem Cell Therapies in Retinal Disorders". Cells. 6 (1): 4. doi:10.3390/cells6010004. PMC 5371869Freely accessible. PMID 28157165. 
  9. ^ "Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation : Protocol Exchange". www.nature.com. Retrieved 2017-05-25. 
  10. ^ Haan, A. Dénise den; Veldkamp, Marieke W.; Bakker, Diane; Boink, Geert J. J.; Janssen, Rob B.; Bakker, Jacques M. T. de; Tan, Hanno L. (2013-03-13). "Organ Explant Culture of Neonatal Rat Ventricles: A New Model to Study Gene and Cell Therapy". PLOS ONE. 8 (3): e59290. doi:10.1371/journal.pone.0059290. ISSN 1932-6203. PMC 3596330Freely accessible. PMID 23516623.