Prostaglandin actions in established insect cell lines

The Insect Cell Culture Working Group at USDA, ARS, BCIRL. From left: Yao-Fa Li*, Hongwei Zhang, David Stanley (with Watson), Yiyun Xu, Joseph Ringbauer, Jr., Kaile Zhou, Steve Saathoff, Cynthia Goodman, and Tamra Reall Lincoln. *On sabbatical leave from Plant Protection Institute, Hebei Academy of Agricultural and Forestry Sciences, Baoding, China.

Prostaglandins (PGs) are derived from arachidonic acid and two other C20 polyunsaturated fatty acids. They are most understood in the biomedical arena, where they exert many actions, such as proinflammatory reactions to infection and wounding. The biological significance of PGs in insects and other invertebrates has come to light over the last 30 years. Among their actions, they release egg-laying behavior in newly-mated crickets and mediate many aspects of insect immunity. More recently, we have been investigating possible PG actions in established insect cell lines. Treating cell lines with various PGs leads to substantial changes in gene and protein expression, from which we inferred that PGs are present and exert biologically meaningful actions in insect cells. The identity of these biological actions remains obscure. In the current article, we investigated whether PGs influence cell proliferation or viability of insect cell lines. We treated cell lines with PGs and also by inhibiting PG biosynthesis using pharmaceutical agents. We used lines from three insect orders: Coleoptera (red flour beetle, Tribolium castaneum; Goodman et al., 2012, In Vitro Cell Dev Biol – Animal 48:426), Hemiptera (squash bug, Anasa tristis; Goodman et al., 2017, this issue) and Lepidoptera (tobacco budworm, Heliothis virescens; McIntosh et al., 1981, In Vitro 17:649). At low doses, the added PGs did not influence cell numbers; at higher doses, they led to decreased cell numbers in all three lines. We used flow cytometry to determine the decreased numbers are due to cell death, rather than to reduced cell proliferation. PGs are identified based on numbers of double bonds and functional groups attached to their 5-membered rings. PGDs, PGEs, and PGFs are the classical PGs that act via cell surface receptors and PGAs are cyclopentenone PGs that enter cells and act by forming covalent bonds with cysteine residues in cellular proteins. PGA1 and PGA2 treatments influenced cell numbers more strongly than PGE1 or PGE2. Inhibiting PG biosynthesis also led to reduced cell numbers. We infer that PGs, like many biochemicals, are Janus-faced molecules, necessary for cell functions when present in appropriate concentrations and deleterious when present at concentrations outside the narrow, necessary range. The influence of PGs and inhibitors of PG biosynthesis on cell numbers differed among the three cell lines. Overall, our studies confirm insect cells require PGs to mediate their on-going homeostatic physiology.

Yao-Fa Li, Hongwei Zhang, Joseph A. Ringbauer, Jr, Cynthia L. Goodman, Tamra Reall Lincoln, Kaile Zhou, and David Stanley. Prostaglandin actions in established insect cell lines. In Vitro Cellular & Developmental Biology–Animal 53: 421–429, 2017.

Identifying Connexin Expression and Determining Gap Junction Intercellular Communication in Rainbow Trout Cells

From left: Joshua Hooper (Performed work as a 4th year thesis student, currently in medical school at the University of Toronto), Dr. Stephanie DeWitte-Orr (Associate Professor, Wilfrid Laurier University, Waterloo, ON) and Sarah Poynter (PhD candidate in the DeWitte-Orr lab).

Gap junctions are groups of membrane-bound channels that allow the passage of small molecules and ions between cells, permitting cell-cell communication.  These channels play a key role in cell homeostasis, development, and electrical coupling. Three common rainbow trout cell lines, RTgill-W1, RTgutGC, and RTG-2, have been used extensively in toxicological and virological assays; however, there has not yet been a study exploring gap junction presence or function in any of these cell lines. Gap junction presence was determined by screening for gap junction protein alpha 7 and alpha 1 (GJA7 and GJA1) presence at the transcript level and GJA7 at the protein level; and all three cell lines expressed these connexins at the transcript and protein level.  Gap junction function was determined using Lucifer yellow dye migration with the scrape and load technique; a first for this technique in fish cells.  Lucifer yellow dye migration was quantified in the presence and absence of a gap junction inhibitor, phorbol 12-myristate 13-acetate (PMA).  RTG-2 and RTgill-W1 cells showed significant dye migration that was inhibited by PMA while RTgutGC did not. The importance of length of serum deprivation was also highlighted in this study; 24-h serum deprivation resulted in greater dye migration compared with 30-min serum deprivation.  Future researchers using this technique will benefit from the understanding the importance of optimizing the length of serum deprivation prior to gap junction function quantification. Taken together, our study shows that rainbow trout cells express connexin transcripts and proteins, and that RTG-2 and, to a lesser extent, RTgill-W1 cells possess functional gap junctions.

Joshua Hooper, Sarah J Poynter, Stephanie J DeWitte-Orr. Identifying connexin expression and determining gap junction intercellular communication in rainbow trout cells.  In Vitro Cellular & Developmental Biology–Animal 53: 406–416, 2017.


Morphogenesis and histology of cultures of Iris ensata Thunb. generative organs.

Lydmila I. Tikhomirova

The morphogenetic study of plants is a complicated task in biology, yet it has become an increasingly intense area of research. Practical areas such as meristem technologies, embryo culture, haploid technologies, cell breeding, gene and cell engineering are actively developing and require fundamental knowledge and research, including the study of mechanisms of plant development in tissue culture. Despite all the experiments on morphogenesis and regeneration of plants in tissue culture, the systems of in vitro propagation for I. ensata are still poorly developed. This is due to the lack of clear, reproducible methods, their complexity, poor knowledge on the morphogenetic potential of organs and tissues, and on the ways for morphogenetic control of this culture. The aim of the present research was to determine the shoot regeneration process based on the anatomical structure of the explants used and the composition of culture medium. The process of I. ensata in vitro morphogenesis depends on many factors. The use of exogenous hormones is one of the main approaches to obtain regenerated plants. In this study, the proper choice of explants was crucial. It was determined that the organs containing meristem tissue and with morphogenetic capacity should be used as explants. The use of the floral organ fragments as the primary explants was optimal for I. ensata at the initiation stage. The direct regeneration in the explant tissue of the rachis and perianth tube resulted in the formation of shoots typical of this species. The shoots were developed from meristems preexisting in the explants. Meristematic activity was not observed from anatomical structures of the ovary, style and filaments as well as the explants of these organs during the entire period of cultivation (30 d). This result likely explains the lack of regenerative capacity of these explants.

Lydmila I. Tikhomirova. Morphogenesis and histology of cultures of Iris ensata Thunb. generative organs.  In Vitro Cellular & Developmental Biology-Plant 53:270-277, 2017

Three new terms for in vitro biology: invitromatics, invitromes, and invitroomics

From left to right:  Lucy E.J. Lee, Phuc H. Pham, Niels C. Bols, and Vivien R. Dayeh

We have introduced in a recent edition of In Vitro Cellular & Developmental Biology Animal three new terms for in vitro biology: invitromatics, invitrome, and invitroomics (Bols et al., 2017).  The genesis of these terms was at the 14th International Conference on Invertebrate and Fish Cell Culture meeting in San Diego, CA in June of 2016 when the authors gave an overview on the availability of fish cells now and in the future. The terms were illustrated in our paper with the cell lines of rainbow trout.  Invitromatics is the science and history of establishing, characterizing, engineering, storing, and distributing cell lines.  Rainbow trout invitromatics began in 1962 with the development of fibroblastic cell line, RTG-2, by Wolf and Quimby (1962). The invitrome is the cataloging of cell lines around a common theme. The theme is the choice of the researcher so RTG-2 belongs to many invitromes. For example, RTG-2 belongs to the fibroblast invitrome.  Another cataloging is based on the species or animal group.  RTG-2 belongs to the rainbow trout invitrome and the fish invitrome. The rainbow trout invitrome has approximately 54 cell lines, and according to Cellosaurus, an online reference site, there are 530 fish cell lines (Bairoch, 2017).  The availability of some of these cell lines is questionable and has led to the term zombie invitrome.  Zombie cell lines are cell lines that have been described in the literature but not placed in repositories and not used in further publications.  However, they might reside in a liquid nitrogen dewar somewhere ready to rise again (see picture of authors with their possible zombie zoos).   Invitroomics is the application of cell lines to study the cellular and molecular biology of multicellular organisms or to manufacture useful products.  In other words the use of invitromes is invitroomics.  We illustrated this for viral invitroomics by reviewing all the studies on viruses in which rainbow trout cell lines have been employed.  Approximately 62 viruses in 15 viral families have been studied with the rainbow trout invitrome.  The use of cell line(s) to study a specific virus and disease might be designated by putting the name of the virus in front of invitroomics.  For example, salmonid alpha virus (SAV) causes pancreas disease (PD), and all the studies that utilize cell lines to investigate SAV and PD could be referred to as being SAV invitroomics. Whether biology really needs yet more terms revolving around ‘omics’ is an uncertain proposition but we hope so.  The terms might be considered a rebranding of cell lines.  Rather than focusing on the negative aspects of a particular cell line, we are trying to illustrate that the power of cell lines might be in collectively what they can tell us about a species, organ, tissue or process.  We encourage others to write reviews on the cell lines of their interest, using the organization of invitromatics, invitromes and invitroomics.

Niels C. Bols, Phuc H. Pham, Vivian R. Dayeh, Lucy E.J. LeeInvitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout. In Vitro Cellular & Developmental Biology-Animal, 53:383-405, 2017.

Bairoch A (2017) Cellosaurus a knowledge resource of cell lines. version 22 (May 2017).

Bols NC, Pham PH, Dayeh VR, Lee LEJ  (2017)  Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout.  In Vitro Cell. Dev. Biol. – Animal 53: 383-405.

Wolf K,  Quimby MC (1962) Established eurythermic line of fish cells in vitro. Science 135: 1065-1066.