Sponges are one of the most prolific sources of marine natural products (MNPs) [1
]. Unfortunately, many pharmaceutically relevant MNPs are found only in trace amounts within the source sponge [2
], and it is neither economically nor ecologically feasible to harvest enough wild sponge biomass to supply the necessary quantities for clinical drug development and manufacture [3
]. In situ aquaculture of whole sponges or sponge “explants” (fragments) has been successful in situ for a limited number of species [4
], however, the inability to control environmental conditions (e.g., extreme weather events, harmful algal blooms, etc.) makes in situ aquaculture a less desirable biological option.
In vitro cultivation of sponge cells is another biological option for production of biomass or bioactive metabolites [5
]. Due to their cellular organization, sponges can be dissociated into cells that will reaggregate and differentiate to form a functional sponge [6
]. Cell culture allows for precise control of environmental variables and selection or optimization of conditions that favor increased production of biomass and/or bioactive metabolites. Typically, normal (mammalian) cells form a monolayer and remain attached to the substrate to proliferate. Increased understanding of basic metabolic processes at the cellular level in mammalian cell cultures has led to a transition to understanding these processes in differentiated, three-dimensional (3-D) populations of cells [7
]. Cells in two-dimensional (2-D) culture exhibit different morphological and physiological characteristics, such as changes in functionality, morphology, phenotype, and metabolic activity [7
]. Cell-to-cell and cell-to-extracellular matrix (ECM) interactions play a key role in these characteristics and are limited in 2-D culture [7
While there is still much to learn about basic cellular and molecular processes in sponge cells, there is much to gain from research using sponge cell and “tissue” cultures, e.g., primmorphs [8
]. The aim of this study was to evaluate sponge cells cultured in three 3-D substrates: FibraCel®
disks, thin hydrogel layers, and gel microdroplets (GMDs), with the goal of applying one or more of these methods to scale-up production of sponge biomass and/or bioactive metabolites.
disks (Eppendorf, Enfield, CT, USA) are solid support matrices made of a nonwoven polyester mesh and a polypropylene support grid that are treated to attract cells to attach to the disk [9
]. The porous nature of the nonwoven mesh increases the surface area available for cell attachment because the cells can infiltrate the mesh and attach on the inner surfaces as well as the outside of the disk [10
disks are designed for use in cell culture bioreactors, which allows for long-term high density cell cultures in perfusion mode. This is particularly advantageous for the production of biologically derived compounds. We have previously demonstrated that cells of the sponge Axinella corrugata
could be immobilized on FibraCel®
disks, and that the cultured cells continued to produce stevensine [11
Hydrogels, like gelatin and agarose, are crosslinked polymer networks with high water content [12
]. To form a hydrogel, a liquid precursor solution seeded with living cells is polymerized using physical or chemical crosslinking [12
]. The gels imitate elements of native ECMs, including exhibiting mechanics similar to soft tissues and promoting cellular adhesion. These properties increase the appeal of hydrogels as a surrogate ECM [12
The properties of hydrogels are translatable to GMDs, which are created using the same materials (gelatin, agarose). GMDs are small spheres of hydrogel in which few or even single cells can be encapsulated [12
]. The small volume of GMDs makes them highly permeable, which allows for communication among cells and the diffusion of cellular metabolites [12
]. Culturing marine sponge cells presents some unique challenges, for example, marine sponges require high salinities which can prevent the hydrogels from solidifying properly. In addition, many hydrogels require curing periods at high temperatures that are lethal to sponge cells.
Researchers have made extensive efforts to create a sponge cell line [3
], but until recently only primary cell cultures with a finite lifespan have been established. With the development of an improved nutrient medium [14
], Conkling et al. [15
] demonstrated exceptionally rapid rates of cell division in several species of sponges and developed finite cell lines for three species of the genus Geodia
. These recent advancements had not yet been applied to 3-D sponge cell culture. We hypothesized that combining these methods, i.e., improved nutrient media and the use of 3-D matrices, to obtain rapidly dividing cells in a 3-D system would increase cell-to-cell and cell-to-ECM communication and would be more similar to how sponges function in nature, including the production of bioactive compounds.
Each 3-D method presents unique combinations of advantages and weaknesses (Table 5
The major limitation of FibraCel®
disks is the inability to quantify cells. The most direct way to determine cell concentration is by counting cells recovered from the 3-D matrix. Complete removal of cells from FibraCel®
disks was not possible. The cells could not be removed from the mesh using CMF, which is a widely accepted method of disaggregating sponge cells. Cell recovery was not a problem with the other treatments because the ULTA hydrogel matrix itself could be digested enzymatically. Any agent capable of digesting the polyester FibraCel®
mesh would have also destroyed the cells. In addition, the fibers of the mesh obscured cells and aggregates within the disks, which made accurately counting cells difficult. As an alternative, total protein analysis was attempted as a proxy to determine an increase in cell numbers. As previously reported [15
], the cells become pigmented when cultured in M1 medium, and the pigment interferes with protein measurements, which are based on light wavelength, rendering the total protein analysis assay useless for quantifying protein from these cells. An increase in overall pigmentation of the FibraCel®
disks using colorimeter software as a proxy for quantitative data suggested an increase in cell concentration, however, these data are qualitative. In conclusion, FibraCel®
disks are not recommended for any sponge cell culture applications using M1 medium that require precise counts of cell concentration. The hardiness of these disks and their ability to retain cells, however, make them a promising candidate for production of sponge biomass via aquaculture. FibraCel®
disks can be seeded with cells and could be transplanted to an aquaculture system for scale-up of biomass. FibraCel®
disks are also ideal candidates for production of bioactive compounds in vitro. They are designed for use in a packed-bed bioreactor, so a large quantity of cells can be cultured, and the desired natural products can be removed with the spent medium or harvested cell biomass [11
]. This process is designed to be scaled-up. In addition, the process of seeding FibraCel®
disks with cells requires minimal effort and the disks themselves are commercially available and inexpensive.
Like FibraCel® disks, cells cultured in ULTA thin hydrogel layers may be transplanted to a land-based aquaculture system for scale-up of biomass production. However, the ULTA hydrogel matrix is more delicate than FibraCel® disks and would need to be placed in aquaculture tanks or raceways with gentle water movement. Although the ULTA thin hydrogel layers did not dissolve throughout the culture period, as was observed with the porcine gelatin layers, the matrix will degrade over time. This is a potentially limiting factor for aquaculture purposes, as the gel may not remain intact until the sponge cultures have stabilized and can be attached to a more robust substrate. The advantage of using ULTA thin hydrogel layers in vitro is the ability to monitor individual cells and aggregates that are immobilized in place over an extended period of time. Due to the clarity of the ULTA hydrogel, layers of cells can be microscopically imaged, and the individual layers can be stacked and analyzed three-dimensionally using software such as Image J. This feature can be especially useful for studying sponge cell differentiation and the formation of adult sponge architecture. The ULTA thin hydrogel layer method is more labor intensive than the FibraCel® method: temperature and time requirements for forming the ULTA thin hydrogel layers must be balanced with the temperature limits of sponge cells, which lose viability when exposed to temperatures above 37 °C.
Many of the same characteristics of the ULTA thin hydrogel layers are translatable to GMDs, which are made of the same material (ULTA). As the ULTA GMDs are delicate and degrade over time, they are recommended for in vitro research. For example, sponge cells cultured in ULTA GMDs could be applied to in vitro production of MNPs, due to the rapid diffusion of medium and sponge products into and out of the matrix and to the ability to culture and scale-up the droplets in spinner flasks. ULTA GMDs would be useful for studying sponge cell metabolism for the same reasons. This method may also be used to create GMDs with single cells by serial dilution to study cell division, differentiation, and formation of 3-D architecture in the GMD small-volume microenvironment using high content imaging and/or flow cytometric analyses. The formation of ULTA GMDs is the most labor intensive of the methods evaluated. Therefore, scale-up may be challenging. Future studies using GMDs will require the development of an automated method to form the droplets, which will increase the consistency of droplet size and decrease the time and effort to create the GMDs.
Shared trends in cell concentration over time in all methods indicate nutrient limitation. The initial rapid increase of cells appears to exceed the carrying capacity of the medium, resulting in a decrease and then slow increase towards a plateau at a cell concentration that can be supported by the medium. This is further supported by the fact that all 3-D cultures reached similar final cell concentrations (3.26–8.55 × 107). Further studies using perfusion culture, in which fresh medium is constantly added to the culture vessel while spent medium is removed, may enable a greater increase in cell numbers.
Before moving forward with further research using the original M1 or modified versions of M1 medium, it will be necessary to understand what is causing the pigmentation in cells cultured with this medium. In addition to interfering with light wavelength-based assays and measurements, the pigmentation also interferes with the ability to observe the cells or their components with fluorescent dyes. The inability to use these assays severely limits the ways in which marine sponge cells can be studied and the questions researchers can investigate. This pigmentation has been observed in a number of sponge species, including three species belonging to the genus Geodia
]. Conkling et al. [15
] hypothesized that this pigmentation is caused by the increased production of melanin, possibly due to some components of M1 medium.
It is important to note that M1 medium was first optimized for a different sponge species (Dysidea etheria
) and only for short term (48 h) culture [14
]. Additional optimization of M1 medium for long term culture may also be beneficial in inducing cell differentiation. An optimized version of the M1 medium (OpM1) [16
] contains various growth factors, vitamins, and fetal bovine serum and has been shown to increase the maximum cell density and number of cell population doublings in 2-D cultures of the related species Geodia barretti
]. Combining this medium with 3-D culture methods for G. neptuni
has yet to be attempted and may produce favorable results.
Cells from the marine sponge Geodia neptuni were successfully cultured using three 3-D culture methods: FibraCel® disks, ULTA thin hydrogel layers, and ULTA GMDs. These cultures performed comparably to 2-D control treatments, and there are merits to each culture type that recommend them for various applications. No cell differentiation was observed in any culture treatment, and further research is required to induce differentiation and sponge architecture formation.
The cause of the pigmentation observed when sponge cells are cultured in M1 medium needs to be addressed, and if possible, mitigated to prevent its interference in light-based analyses. This would broaden our ability to collect data on marine sponge cells and expand the types of studies that can be conducted using medium M1 and its derivatives, which are to date the only nutrient media capable of inducing cell division in marine sponge cells.
Continued research using the 3-D methods detailed here should focus on perfusion cultures to determine whether the cultures are nutrient limited. Finally, further research on scaling up these methods is recommended to increase their usefulness for application to production of sponge-derived chemicals with human health applications.