Primary cell culture from embryos of the common house spider Parasteatoda tepidariorum

doi:10.21203/rs.3.rs-5312272/v1

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Primary cell culture from embryos of the common house spider Parasteatoda tepidariorum

https://doi.org/10.21203/rs.3.rs-5312272/v1

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Background

Spiders have emerged as valuable models in evolutionary developmental biology, but primary cell cultures from spider embryonic tissues have not been fully explored. In this study, we describe the first successful long-term cultivation of embryonic cells from the common house spider, Parasteatoda tepidariorum. We initiated five independent primary cultures using mechanical and enzymatic dissociation methods, comparing two culture media, Leibovitz’s L-15 and Grace’s Insect Medium, under varying pH conditions.

Results

Cultures exhibited diverse cell morphologies, including round cells in suspension and elongated, neuron-like cells. The most successful culture, initiated with Grace’s medium at pH 7, was passaged four times and maintained for over six months. We also tested collagen type I-coated wells to improve cell adhesion. Our results indicate that P. tepidariorum embryonic cells proliferate better at pH 7, and Grace’s medium supports long-term growth, while L-15 promotes more cell differentiation.

Conclusions

This culture system provides a valuable platform for functional genomics studies, with potential applications in evolutionary and developmental biology research.

spiders

embryos

cell culture

cell type.

Spiders have evolved remarkable novelties, such as venom and silk, making them a focus of research for their toxins [1, 2] and the biomechanical applications of their silk threads [3, 4]. Spiders have also become powerful model organisms in evolutionary developmental biology, particularly the common house spider Parasteatoda tepidariorum [5–7]. Several practical advantages make P. tepidariorum a valuable model, including ease of culture, a short life cycle, year-round access to embryonic stages, and a growing range of genetic and molecular resources [6, 8, 9]. P. tepidariorum has provided insights into fundamental developmental processes, such as axis formation and segmentation (summarized in [6, 7]), as well as the genetic basis of gut formation [10] and eye development [11, 12]. Traditionally, these studies have focused on investigating candidate genes using knockdown techniques such as parental and embryonic RNA interference. More recently, single-cell RNA-sequencing of embryos at different developmental stages has expanded our understanding of cellular diversity, organ development, and embryogenesis [13–16].

Despite these advances, the development of primary cell cultures from P. tepidariorum specifically, and from spiders in general, remains an unexplored avenue. In vitro systems offer experimental possibilities for functional studies, enabling precise genomic manipulation to investigate embryogenesis at the cellular level [17], cell differentiation [18], biochemical pathways and the effects of compounds such as hormones [19, 20]. This report describes the initiation and maintenance of primary cell cultures from embryonic tissues of P. tepidariorum.

Inbred individuals of P. tepidariorum originate from our laboratory culture that was founded with spiders from Göttingen (Germany). The spiders were kept at 28°C with 60% humidity on a gradual light/dark cycle of 16/8 hours. Adults were housed individually in plastic vials and fed twice a week with laboratory colonies of Musca domestica flies. Cocoons were transferred into Petri dishes and opened gently with forceps to expose the eggs. Embryos were collected after four days, corresponding to developmental stages 10–12 according to [21]. For each experiment, 20–30 eggs were used.

The eggs were dechorionated by immersion in 3% bleach for 2 minutes, followed by rinsing 5 times with running Milli-Q^® water (Millipore). The embryos were then submerged in 70% ethanol for 2 minutes and rinse 4 times with sterile Milli-Q^Ⓡ water. Embryonic tissues were mechanically dissociated using a plastic pestle in 100 µl of complete medium (see below). The suspension was directly transferred into a well of 48-well tissue culture-treated plate (Corning®), and the complete medium topped up to a total volume of 300 µl. The plate was sealed with tape to minimise evaporation.

We also tested enzymatic digestion using 0.25% trypsin from bovine pancreas (Sigma-Aldrich®) in a Ca- and Mg-free saline solution containing 0.2% EDTA [22]. Embryos were incubated in this solution on a gentle shaker for 30 minutes at room temperature. Following incubation, any remaining undigested tissue was further dissociated by applying gentle strokes with a plastic pestle. After dissociation, the embryonic tissue was rinsed three times with complete medium by centrifugation at 400 g for 5 minutes at room temperature. The supernatant was removed, the cell pellet was resuspended in fresh complete medium, and the cell suspension was added to a well of a 48-well plate as above.

We tested two culture media and different conditions (Table 1): Leibovitz’s L-15 GlutaMAX™ (Gibco™) supplemented with 10% tryptose phosphate broth (Gibco™), commonly used for tick cell culture [23, 24], and Grace’s supplemented Insect medium (Gibco™). Both media were supplemented with a solution of 100 U/ml Penicillin and 100 µg/ml Streptomycin (Gibco™), 10% or 20% fetal bovine serum (FBS) (Gibco™), and 10 µM ROCK inhibitor Y-27632 (AbMole), the latter only for the first week of culture. Different pH conditions (i.e., 6 and 7) were tested (Table 1), and pH adjustments were made with 1N NaOH and maintained using 0.1 M BES buffer (Millipore).

Table 1

Summary of primary cell cultures of *P. tepidariorum* embryos initiated in this study.
Code	Date	Dissociation	Medium	FBS	pH	Duration
PTE1	02/04/2024	Enzymatic	Grace	10%	6	7 weeks
PTE2	16/04/2024	Enzymatic	Grace	10%	6	8 days
PTE3	29/04/2024	Mechanical	Grace	10%, 20%	6,7	6 months, 4 passages
PTE4	01/05/2024	Mechanical	Grace	20%	6	30 days
PTE5	05/06/2024	Mechanical	L-15	10%	7	2 months

Cells were incubated at 28°C without CO₂ in a humidified incubator, and the medium was not replaced for the first 14 days; afterwards, the medium was refreshed weekly. Since most cells were in suspension, the old medium was transferred to a new well, and fresh medium was added, effectively performing sub-passages. If we observed cell proliferation in the subpassages, the cultures were continued and eventually pooled to increase density. A full passage was performed by splitting the cells into two wells. Cultures were regularly monitored for cell growth and contamination using a Leica DMi1 (20X magnification) or Zeiss Axio observer equipped with an Axiocam ERC 5s for 40X and 63X magnification.

Because some cells were only loosely attached to the tissue culture-treated plates, we attempted to enhance attachment by coating a well with collagen type I High Concentration, rat tail (Corning®). The coating was performed by covering the well with collagen I which was incubated overnight at 4°C, and then air dried at room temperature under laminar flow cabinet before seeding.

Five independent primary cultures (PTE1-5) from P. tepidariorum embryonic tissues were initiated in this study (Table 1). Most cultures were lost due to contamination, or they did not show substantial growth, except for culture PTE3 started on 29/04/24. This culture was fully passaged four times and used to test different pH conditions, as well as the effect of collagen type I coating. At time of writing, this culture is six months old.

Both enzymatic and mechanical dissociation produced comparable results, but mechanical dissociation was preferred. It required fewer washing steps, reducing the risk of cell loss. Additionally, as there were no washing steps, nutrients and growth factors from the eggs remained in the culture, potentially stimulating cell proliferation. Immediately after seeding, the suspension consisted of round cells of varying sizes and cell aggregates.

PTE5 with L-15 medium produced the most diverse cell types. After just one day in culture, we observed small round cells in suspension, and adherent elongated cells (Fig. 1A). The elongated cells were also observed migrating from a tissue clump (Fig. 1B). By day five, the elongated cells became filled with vesicles (Fig. 1C) which increased in number until day eight, after which the vesicles disappeared, probably secreted into the medium. These elongated cells displayed a range of morphologies, including oval and triangular shapes (Fig. 1C), and seemed to be interconnected by protusion extending out of the cell body (Fig. 1C and Additional file 1: Fig. S1), some of which were particularly long resembling neuronal processes (Fig. 1D).

On day six, we observed clusters of large vacuolated cells, either transparent or filled with vesicles (Fig. 1E), alongside other cells in suspension with varied morphologies (Fig. 1F). After one month, the culture predominately consisted of loosely attached, round cells and some occasional elongated cells (Additional file 2: Fig. S2). Cell growth in L-15 medium was limited, and the culture was eventually interrupted.

In contrast, trials using Grace’s insect medium were more successful, although the cells did not differentiate as much as in L-15 (Fig. 2). After one day, we observed small round cells in suspension and cell aggregates, likely from partially dissociated tissue (Fig. 2A), but no elongated cells as observed in L-15. The round cells generally proliferated, increased in size, and were either in suspension or loosely attached, sometimes growing on top of each other (Fig. 2B). The most successful culture was PTE3, which was fully passaged four times, and is still currently in culture. After two weeks we performed the first full passage by splitting PTE3 into two wells to test in parallel pH 6, which is the normal pH for Grace’s medium, and pH 7. In both cases, we observed proliferation of the round cells, but interestingly, we observed elongated cells only in pH 7. The elongated cells in Grace’s medium seemed smaller and did not produce long protusions as seen in L-15 (Fig. 2B-C). Since we did not use trypsin to detach the cells for passaging, subsequent passages resulted in a monoculture of round cells in suspension, although they had different sizes, with only occasional small elongated cells observed (Fig. 2C).

Cell growth in Grace’s medium was more successful than in L-15. After a few days post-plating, exponential proliferation occurred, particularly at pH 7. Cells at pH 7 also appeared larger, prompting us to convert all cultures to pH 7. After the initial growth peak, cell proliferation was slow, although it continued throughout the study, and enough to perform a total of four full passages in six months.

Lastly, we used the subpassages to test whether cells could attach to wells coated with collagen type I. Indeed, cells adhered to the collagen filaments and grew preferentially on the collagen-coated areas of the well (Fig. 2D). Under these adhesion conditions, we did not observe substantial growth; however, this may have been due to insufficient initial cell density.

In this study, we report, for the first time, the successful long-term cultivation of embryonic cells from a spider species, P. tepidariorum. The only other similar study in the literature was on embryos of the Brazilian armed spider Phoneutria nigriventer [25], but in that study, no cell growth occurred, possibly because the cells were kept at 15°C - a temperature too low for optimal proliferation, especially for a tropical species. In both the P. nigriventer and P. tepidariorum cultures, round cells in suspension were observed, but in the former study adhesion and differentiation were observed.

A critical factor we identified was that cell proliferation may have been hindered by the other authors’, as well as our, practice of changing the medium too frequently. Even though we centrifuged the cells, some were inevitably removed during medium changes, leading to unintentional sub-passaging. The one instance when we did not change the medium for three consecutive weeks coincided with a period of significant cell growth and confluence, allowing us to perform the second full passage. This suggests that frequent medium replacement may negatively impact cell proliferation, explaining the slow growth after the initial surge. Infrequent medium changes have been used successfully when generating tick primary embryo-derived cell cultures from small numbers of eggs [23]; moreover, tick cell cultures are known to exhibit slow rates of multiplication, especially during early stages [23, 26, 27].

Another key observation was that spider cells seem to thrive better at pH around 7, similar to mammalian cells, unlike insect cells, which are typically cultured at lower pH values, around 6.3 [28]. This is consistent with reports that the haemolymph of various spider species has pH between 7.48 and 8.21 [29]. For future experiments, adjusting the pH to around 7.5 may further enhance cell proliferation and viability.

Upon initial plating, we consistently observed the proliferation of small, round cells, which later became larger. In Grace’s medium, these cells continued to proliferate, while in L-15, we saw a greater degree of differentiation, but slower growth. This distinction between the two media suggests that they could be used complementarily, with Grace’s medium promoting proliferation and L-15 driving differentiation.

Our cultures were initiated from embryos at developmental stages in which organ differentiation had already begun [21]. Future studies could explore the establishment of cultures from earlier embryonic stages, which might yield multipotent cells with broader differentiation potential.

Given that P. tepidariorum is easy to maintain in the lab and produces a high number of cocoons [7], initiating primary cell cultures from embryos at various developmental stages should be feasible for any laboratory equipped with basic tissue culture facilities, such as a laminar flow hood and incubator. Gene knockdown through RNA interference (RNAi), either parental or embryonic, is already well established in this spider species [6, 7]. The application of RNAi in cell culture should be very straightforward and could open the door for large-scale functional experiments. Additionally, more complex gene-editing methods like CRISPR-Cas, which are often technically demanding in vivo, may prove more feasible in an in vitro setting. Moreover, the use of transfection for gene knock-in, which has already been demonstrated in tick cell cultures [30–32] and is well-established in insect cell cultures [28], could open up new avenues for genetic experimentation in spiders.

In conclusion, we have presented the first detailed description of a spider cell culture derived from embryonic tissues and successfully established a long-term cell culture. This system offers a valuable and accessible platform for studying cell differentiation, developmental pathways, and biochemical signalling in a controlled environment, therefore contributing to research advancements in arthropods’ developmental biology.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data and materials are included in this article and its Supplementary Information.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Swiss National Science Foundation COST grant IZCOZ0_205460 to G.Z.

Authors' contributions

G.Z. designed the project and wrote the manuscript. A.H. performed the experiments and documented the data. All authors read and approved the final version of the manuscript.

Acknowledgements

We thank Alexandre Reymond for providing access to his cell culture laboratory, and Lesley Bell-Sakyi for her insightful advice regarding cell culture conditions and invaluable feedback on the initial manuscript.

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No competing interests reported.

FigS1.png
Supplementary Information Additional file 1: Fig. S1. Neuron-like cells in L-15 medium after six days of plating. The elongated cells are filled with vesicles and have extensions connecting the cells to each other. Live, phase-contrast inverted microscope, 20X magnification.
FigS2.pdf
Additional file 2: Fig. S2. Culture in L-15 medium after one month (A-B) and two months (C-D). No cell proliferation nor further differentiation was observed. Live, phase-contrast inverted microscope, 20X magnification.

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Primary cell culture from embryos of the common house spider Parasteatoda tepidariorum

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Abstract

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Background

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