Developments in marine invertebrate primary culture reveal novel cell morphologies in the model bivalve Crassostrea gigas

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Aquatic Biology

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Introduction

Materials and methods

Establishing cell cultures with a large explant method

Buffers and culture materials

Tissue culture vessel coatings

Oyster cell passage

Cell imaging

Quantification of gene expression

DNA Barcoding of cell cultures

Results

Discussion

Conclusions

Supplemental Information

Supplementary methods for dissociation of oyster and changing culture conditions that were not used to generate any of the figures, but are relevant and interesting experiments that have not been reported previously

DOI: 10.7717/peerj.9180/supp-1

Dissected oyster heart contractions

Heart removed from an oyster clearing contracting rhythmically 6 weeks after dissection. Heart is approximately 3mm across.

DOI: 10.7717/peerj.9180/supp-2

Live cell imaging of oyster heart cells

Live cell imaging of Pacific oyster heart culture 1 day post establishment, showing fibroblast-like cells and hemocytes adhering and moving across tissue culture plastic. Scale bar = 900 μm. Real time is displayed top left.

DOI: 10.7717/peerj.9180/supp-3

CO1 barcoding sequences from oyster cell cultures

DOI: 10.7717/peerj.9180/supp-4

Raw data from qPCR of oyster cell cultures—used for Fig. 4

DOI: 10.7717/peerj.9180/supp-5

Pacific oyster anatomy diagram

Anatomy of the pacific oyster with labels for all major tissues visible. Digestive gland (not visible) is found enveloped by gonadal tissue. Top (right) layer of mantle has been removed.

DOI: 10.7717/peerj.9180/supp-6

Anatomy of oyster

(Provided to give an original)

DOI: 10.7717/peerj.9180/supp-7

Multiple cell morphologies from oyster heart primary culture. Part 1 of 4—fibroblast-like cells

Heart primary culture clearly showing fibroblast-like cells dissociating from aggregations of rounded cells and adhering to culture plastics.

DOI: 10.7717/peerj.9180/supp-8

Multiple cell morphologies from oyster heart primary culture. Part 2 of 4—highly confluent cells

High confluency heart primary culture 28 days after establishment with round, spindle shaped and epithelial-like cells all visible.

DOI: 10.7717/peerj.9180/supp-9

Multiple cell morphologies from oyster heart primary culture. Part 3 of 4—hemocytes surrounding explant

Heart primary culture 12 days post establishment with confluent granulocytes, with round and epithelial-like cells also visible.

DOI: 10.7717/peerj.9180/supp-10

Multiple cell morphologies from oyster heart primary culture. Part 4 of 4—large elongated cells

Large elongated cardiomyocyte like cells. Hemocytes, round, spindle shaped and epithelial-like cells are also visible.

DOI: 10.7717/peerj.9180/supp-11

Oyster primary cell cultures established from large explants of multiple tissues. Part 1 of 4—mantle primary culture

Mantle primary culture showing epithelial-like cells dissociating from aggregations of rounded cells and adhering to culture plastics, similar to heart cultures.

DOI: 10.7717/peerj.9180/supp-12

Oyster primary cell cultures established from large explants of multiple tissues. Part 2 of 4—gonad primary culture

Gonad primary culture with round and fibroblast-like cells visible, 5 days post explant.

DOI: 10.7717/peerj.9180/supp-13

Oyster primary cell cultures established from large explants of multiple tissues. Part 3 of 4—gill primary culture

Gill primary culture with hemocytes, round and fibroblast-like cells.

DOI: 10.7717/peerj.9180/supp-14

Oyster primary cell cultures established from large explants of multiple tissues. Part 4 of 4—adductor muscle primary culture

Muscle primary culture with round, spindle shaped and larger muscle-like cells adhering to the culture plastics.

DOI: 10.7717/peerj.9180/supp-15

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Robert W.A. Potts conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Alejandro P. Gutierrez and Yennifer Cortés-Araya conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Ross D. Houston and Tim P. Bean conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

The sequences are available as a Supplemental File.

Data Availability

The following information was supplied regarding data availability:

All qPCR data and sequences are available in the Supplemental Files.

Funding

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant number BB/M010996/1), BBSRC Institute Strategic Programme Grants (BBS/E/D/20002172 and BBS/E/D/30002275) and Centre for Environment, Fisheries and Aquaculture Science (Cefas) Seedcorn project DP901W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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