Mod3D: A low-cost, flexible modular system of live-cell microscopy chambers and holders.

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

Introduction

Live-cell microscopy imaging has evolved from single chamber observations to large-scale uses in High Content Analysis. Commercial live-cell imaging chambers and plates typically involve injection molded thermoplastic bonded to the glass. However, these chambers are typically expensive relative to 6-, 12-, and 96-well plate formats and plastic dishes, often by an order of magnitude or more, due to the added complexity of a bonded glass surface. Strict manufacturing standards ensure the dishes are flat and are reliably constructed to hold media without leaking. Assembled live-cell chambers are available from commercial sources, but are machined in metal or plastic and are typically very expensive and limited in the scope of use, but are a common choice for perfusion experiments. However, in a typical cell biology laboratory, these live cell chambers can add significant costs.

3D printing methods to manufacture plastic-based items have been historically reliant on expensive commercial industrial printers protected by a series of hardware and software patents. From 2015, open-source software-driven kits and complete printers cut the costs of these devices drastically. This initiative was started at the University of Bath as the replicating rapid prototyper (RepRap) project in 2005 [1], which was released publicly as an open, free license and GNU general public license. This open-source hardware design was coupled to open-sourced Marlin firmware which could translate 3D modeled shapes into G-code machine commands to direct the tool movements in 3D printers. Marlin runs on inexpensive 8-bit Atmel microcontroller chips at the center of the open-source Arduino/Genuino platform [2]. Most early designs were filament deposition modeling, or FDM, in which a plastic filament is forced into a heated extruder at the glass-transition melting point of the material to layer patterns in a Cartesian XYZ space. Mixed orientations of the layer depositions give models high strength in multiple directions of force and are typically flat within 50 micrometers with print resolutions at 100 micrometers or higher, depending on extrusion nozzle size.

Another type of consumer 3D printing, stereolithography (SLA), does not use filaments nor heat, but rather UV-crosslinked resin layers in patterns directed by a <405nm laser line controlled by a mirror and galvanometer. This was further derived to Masked Stereo Lithography (MSLA) by the use of inexpensive light-emitting diode (LED) arrays and consumer electronics-sourced liquid crystal displays (LCD) to replace the galvanometer-directed laser light with a lithography mask created by the LCD in front of an array of 405 nm LEDs. A platform controlled by a stepper motor can then provide resolutions in Z at 25 micrometers, with an Y axis resolution of 1.25 micrometers. For both FDM and MSLA consumer printers, costs have been reduced to under US$300. Thus, subsequent adoption to research labs is becoming increasingly popular for the use of small research-based plastic objects such as racks, holders, brackets, and protein models, but typically not replacing plastic consumables. The cost of scientific laboratory use of injection molded consumable plastics is typically driven by high costs of tool and die manufacturing and the limited markets of potential recovery of those costs. However, these factors are not affected by 3D printing.

Given the resolution and precision of 3D printing, as well as the inherent ability to customize, applications in microscopy are numerous to develop low cost specialized solutions [3, 4] to open sourced projects that significantly lower the cost barrier to high performance microscopy, such as the OpenFlexure system [5-7]. 3D printing has been adopted successfully to create microscopy stage chambers for the study of model organisms in three and four dimensions [8]. Molds have been designed for custom production of silicone-based live cell chambers for multimode and multiplex observations [9]. Given the introduction of these printers to the market, 3D printing is quickly becoming essential to the cell biology or teaching laboratory.

Here we sought to manufacture a lab consumable: live-cell microscopy incubation chambers manufactured by MSLA, bonded to standard 22x22mm glass coverslips, held within universal 96-well format platforms manufactured by FDM, self-assembled with magnets, and designed to be reused. We set out to achieve ease of manufacture, low cost, ability to modify for bespoke applications, reliable containment of media; high dimensional quality, a portion of reusability and reduced environmental impact through the use of minimal/biodegradable plastic. The designs were evolved to be modular, allowing a mix of well sizes and types as opposed to one set format with unused wells. Each design can be used within a single universal 96-well format stage holder, minimizing waste and maximizing experimental flexibility.

Results

The challenges of this project included many (>100) empirical attempts of designs, first using just FDM printing. We attempted to use both polylactic acid (PLA) as well as Polyethylene Terephthalate Glycol (PETG) filaments for 3D printing. We found this method unreliable, with the resolution not up to the standards required, a poor ability of FDM prints to be watertight, and a glass bonding surface with too many potential imperfections to allow reliable bonding. The printing time was also several hours by printing chamber replicates. However, we did find the use of FDM superior for the chamber holder, which we designed on a universal 96-well size format at 4mm thickness with a 20% grid infill to provide a very strong, flat and stiff platform that did not expand or change dimensions up to 37°C (Fig 1A). We settled on a final design that could hold up to three 22x50mm chambers or six 22x22mm chambers (Fig 1B), with the overall dimensions of a universal plastic 96-well dish and a lid made of a frame of FDM plastic bonded to a cut sheet of 0.2mm polystyrene (Fig 1C–1E). Within the top and bottom halves of the holder, two or four 5x1mm neodymium magnets on each half were used to self-assemble the lid onto the base (Fig 1C–1E) and hold the chambers in place to prevent floating under objective pressure while using water or oil immersion objectives. Directional arrows, a logo, and one chamfered corner were incorporated into the design to aid with orientation. Each half of the chamber holders can take 2–2.5hrs to print at standard settings on the printer (200 micrometer layers), but these holders and lids can be reused with a sterilization cycle indefinitely.

Fig 1

Mod3D live-cell microscopy system.

(a) Examples of 22x50 mm 3-chamber holders in the universal 96-well plate format, with MSLA printed chambers, FDM printed holder, inserted magnets and reusable PLA/polystyrene lid. (b) Examples of the 6-chamber holders with different chamber designs. (c-e), Modularity and assembly of the system, where the holders and the lids are reusable.

For the chambers, we switched from using FDM to printing with MSLA resin. This solved two problems: the MSLA prints are essentially one piece of plastic, so leaking was never observed, and the top final surface of the print was extremely high quality for glass bonding. We took advantage of the high print resolution to emboss an alphanumeric labeling of chambers onto the sides to keep track of well identities during experiments. The base design was a 22x50mm chamber that fit two standard 22x22mm #1.5 glass coverslips, with a bonding surface of 1.5mm wide edges, ensuring a reliable bond between the plastic and glass. Within this base template (Fig 2A–2E), we were then able to design multiwell plates up to 32 wells per chamber, or 96 wells total within a single three-position carrier. Chambers were designed so that a 150 micrometer coverslip (#1.5) sat 100 micrometers below the plastic holder to prevent any friction between objective lenses and holder plastic during XY travel.

Fig 2

Examples of varieties of chamber designs in Mod3D.

(a,b) Examples of embossed alphanumeric labels on 4-well (750 μl per well) or 16-well (145 μl per well) chambers. (c) Round single well design (2.8 ml). (d) Example of glass bonding using RTV silicone at the bottom of the chambers. (e) Example of 8-well chambers (260 μl per well). (f) Bridged 2-well chambers in 22x50 mm format, shown bonded to 22x22 mm glass coverslips in (g). (h) Bridged 2-well chambers bonded to a 22x50 mm glass coverslip, with reusable plastic lid.

For bonding the coverslips to the plastic chamber, we previously successfully used room temperature vulcanization (RTV) silicone (Silicone I, General Electric) on manufactured plates in which we milled a hole on the bottom of a 35-mm tissue culture dish [10]. RTV silicone has excellent glass bonding properties, is resistant to all solvents used in cell fixation and permeabilization and can withstand temperatures from -60°C to 200°C. We found two-component silicones cured too quickly for practical handling and Vinyl-terminated Polydimethylsiloxane (PDMS) (Silguard, Dow Chemical) had poor adhesive properties or required priming steps. RTV silicones can be cured by the release of acetic acid or oxime-based curing. Both curing compounds can be toxic to cells, so either a full cure time of 72 hrs was required, or cure time could be accelerated to 16 hrs when left in the high humidity of a tissue culture incubator. The choice of RTV silicone was a combination of cost considerations, ease of use, availability and reliability of bonding. We found the oxime-based curing RTV (SS-433T, Silicone Solutions, OH, USA) to be optimal because of lower viscosity which helps with evening the spread of glue on >16-well chambers. To apply the adhesive evenly and ensure a flat bonded surface, a 30mm diameter soft polymer craft roller was used to spread the glue on a 20x20cm sheet of phenolic resin or glass to generate a thin coat on the roller face, then to transfer this thin layer of glue to the chamber bonding face. Coverslips were then placed on the adhesive surface and pressed evenly with a 3D printed block (S1 Video). We prototyped using clear resin, but found light scattering too problematic for fluorescent microscopy use and switched to semi-opaque black resin. 3D printing also cannot produce optically clear plastic without polishing. Therefore, lids for the chambers were made either by FDM printing the frames and bonding a coverslip glass using RTV silicone, by MSLA printing with a solid top for inverted fluorescence microscopy, or by MSLA printing in clear resin with a 200um thin top surface (Fig 2H). MSLA printing is preferred for the lids to allow sterilization, which is difficult in FDM layered prints. Lids were designed with a gap allowing gas exchange during tissue culture incubation.

During longer-term incubation, beyond 72 hrs, we noted a tendency of early 22x50mm chamber designs on 22x50mm coverslips to warp 0.5mm due to the absorption of humidity by the plastic. This warping was caused by a 1% expansion of the length of the plastic, but not the glass. The design solution for longer-term experiments (days to weeks) was to use a 22x22mm chamber design with a 6-chamber holder (Fig 3). This additionally allowed for greater modular flexibility to match the needs of the experimental setup in regards to the number of wells required. Another alternative was to have two linked 22x22mm chambers glued to two 22x22mm coverslips that fit within the 22x50mm holder format. For longer-term live-cell observations, we designed another holder, where single chambers fit within a Tokai HIT incubated stage (Tokai, Japan). The holder maintains the chamber at the correct height while still allowing a pressure seal to prevent CO2 loss and heat escape (Fig 3). The holder takes advantage of a unique PLA formulation that is thermochromic between 32–45°C. This allows the user a simple visual confirmation of temperature with a transition from grey to orange at 37°C, with a second transition to yellow at 42°C for heat shock experiments (Fig 3B–3D).

Fig 3

Thermochromic chamber holder for live cell incubator stage.

(a) Left to right: the lid, chamber, and holder. (b) Assembly of the chamber and holder in the stage at 25°C with grey color. (c) Placement of the chamber and holder within the incubated temperature and gas control chamber at 37°C with color change to orange. (d) As in panel c, but temperature increased to 42°C (yellow).

As an example of utility, a 22x22 single well chamber was used to culture live human RPE1 cells and subjected to a laser stripe DNA damage assay at 37°C (Fig 4). After 24 hours of culture and transfection, poly(ADP-ribose) polymerase 1 (PARP1) enzyme was visualized accumulating to sites of 405nm induced DNA damage within 25 seconds by an anti-PARP1 chromobody fused to TagRFP and then visualized for an additional 2 minutes (S2 Video).

Fig 4

Sample live cell confocal microscopy data.

RPE1 cell expressing PARP1 chromobody as red fluorescent protein fusion. (a) After 24 hrs growth, immediately after 405nm laser irradiation in the region defined by yellow box. (b) 12 seconds later. Panel c, 25 seconds later, showing PARP1 chromobody recruitment to the site of induced DNA damage. Scale bar is 10um. Timestamp is seconds and milliseconds. 60X oil objective.

To then take advantage of this system and fast prototyping, we designed a perfusion chamber with internal microfluidic passages that could not be created using traditional subtractive manufacturing due to the encapsulated fluid channels. Since MSLA printing has excellent resolution capabilities, we were able to design a microfluidic path in a one-piece print. This model only required inserts of metal piping to connect plastic tubing to a peristaltic pump. Contrary to traditional microscopy perfusion chambers, which typically only have a single inlet and outlet, our design allowed for the even rapid distribution of fluid across the imaging area across a manifold of one into four across the surface of the 22x22mm chamber (Fig 5A). The chambers were used during tissue culture with a removable FDM printed lid bonded with silicone (Fig 5B). However, prior to completing a perfusion experiment, a 22x22mm coverslip was sealed onto the top face of the chamber using silicone vacuum grease (CS16, Silicone Solutions, OH, USA).

Fig 5

Bespoke Mod3D perfusion chamber.

(a) Computer Aided Design (CAD) drawing of microfluidic paths and the 1:4 manifolds. (b) Chamber, culture lid and perfusion lid clamp. (c) A 22x22 mm coverslip is placed on top after cell culture sealed with silicone grease and clamped into place with the PETG clamp. Inlet/outlet aluminum tubing is bonded in place with RTV silicone.

For sterilization, we used a combination of bathing in 70% isopropyl alcohol, bathing in sterile water, and UV irradiation in a HEPA tissue culture hood. Following the bathing steps, the chambers were allowed to dry, then sealed in clear plastic bags. The chambers were then UV sterilized in either a 365nm chamber at 30 mW/cm2 (ELC-500, Electro-Lite) for 10 minutes per side, or in a bespoke 3D-printing curing/sterilization chamber. The curing/sterilization chamber was lined with aluminum foil tape and used an inexpensive (~US$40) 50W 385nm LED array flood lamp (80mW/cm2) (WOWTOU, China) (Fig 6) for at least 30 minutes per side, flipping once to avoid shadows. To ensure sterilization, previously prepared chambers from storage were irradiated with UV once more, and individual wells were washed with PBS and media before plating. This protocol ensured complete sterilization without any need for heat.

Fig 6

Chamber UV sterilization.

(a) 3D printed curing/sterilization chamber lined with aluminum foil tape with a 30W 385 nm LED flood lamp placed on top. (b) Alternate method of chamber sterilization in a UV curing chamber with rotating platform (Electrolite ELC-500). In both methods the chambers are placed in clear polypropylene bags.

We did not observe any obvious growth inhibition, toxicity or morphology effects from chamber growth by TruHD cells [11] either visually or quantitatively with live/dead fluorescence assay (Fig 7A and 7B), we tested for any subtle effects on cell health by analysis of mitochondrial ellipticity, which is a very sensitive measure of cell stress [12]. Even at over 80,000 data points, no significant difference in mitochondrial ellipticity was observed versus commercial chambers (Ibidi uSlide 8 well) (Fig 7C).

Fig 7

Growth and no toxicity or stress in Mod3D chambers.

(A) representative bright field images of cell growth of human TruHD cells. TruHD are growth contact inhibited, non-transformed lines. Scale is 1000um. (B) Fluorescent LIVE/DEAD assay comparing commercial chambers to Mod3D. Panel C, assay of mitochondrial ellipticity using NucBlue and Mitotracker dyes comparing commercial chambers to Mod3D. No significant differences were noted, p<0.5 by simple random sample and a Mann-Whitney nonparametric t-Test.

The modular system resulted in significant cost savings over commercially sourced imaging chambers. 22x22mm single chambers cost 20 cents total: 2 cents in plastic and 18 cents in glass. Double chambers cost 44 cents, with the coverslips contributing to the bulk of the cost. Overall, our Mod3D solution costs were 1/25th of their commercial solution counterparts (Table 1).

Table 1

Cost comparisons of commercially available imaging chambers and the 3D printed microscopy chambers.

Product Name	Retailer	Catalog Number	Description	Approximate Cost Per Chamber ($USD)
Thermo Scientific™ Nunc™ Lab-Tek™ Chamber Slide™	ThermoFisher Scientific	177402PK	25x75mm 8-well polystyrene chamber on soda lime glass microscope slide	14
μ-Slide 8-Well Glass Bottom	Ibidi®	80827	25.5x75.5mm 8-well plastic chamber on Schott glass slide	12
Eppendorf Cell Imaging Slide	Eppendorf	0030742079	26x76mm 8-well plastic chamber on soda lime glass microscope slide	13
8-Well Culture Slides	MatTek Life Sciences	CCS-8	Polystyrene chamber on glass microscope slide	9.3
Mod3D live-cell Microscopy Chamber	N/A	N/A	22x22mm/22x50mm PLA chamber on glass microscope slide	0.20 (22x22 mm)
				0.55 (22x50 mm)

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io: dx.doi.org/10.17504/protocols.io.n92ldz587v5b/v1 and is included for printing as S1 File with this article.

3D printing

Models were designed by Computer Aided Design (CAD) in Fusion360 software (AutoCAD, USA) or the free web-based TinkerCAD software (https://www.tinkercad.com/). 3D models were exported as STereoLithography (.stl) files and used to generate G-code in either Cura 4.11 (Ultimaker) for FDM printing or in Chitubox freeware (https://www.chitubox.com/) for resin printing. For FDM, models were printed at 20% grid infill with a layer height of 0.16 mm on either a Creality Ender 3 or a CR10 printer (Creality, Shenzhen, China). PLA or PETG 1.75mm filament was sourced from 3D Printing Canada (https://3dprintingcanada.com/). For chamber holder tops and bottoms, printing was completed with a >6mm brim on the bottom to ensure the print remained flat. To minimize warping, the print was not removed from the heated print bed until both had cooled to room temperature. Within the print, four neodymium 5x1mm magnets were glued into place on each top and bottom half of the chamber holder with polyurethane or cyanoacrylate glue. Thermochromic PLA filament (TopZel Tritemp Lava) was sourced from TopZeal, China.

For MSLA, an Anycubic Photon printer (Anycubic, Shenzhen, China) was used with eSun PLA BioPhotopolymer resin LC1001 (Shenzhen eSUN Industrial Co., China) using the standard settings for that printer located in the Chitubox profile, with the exception of a 160 second first later exposure and a 30 second per subsequent layer exposure. Each print used black resin and contained four 22x50mm chambers or eight 22x22mm chambers. Chambers and lids were printed directly on the printer platform without any rafts or support. Clear resin was only used for prototyping to confirm microfluidic flow.

Print stereolithography files (STL) are available under free use license on the NIH 3D print server: https://3dprint.nih.gov/discover/3dpx-016293.

Post processing

After printing, 3D prints were released from the platform and washed for 15 minutes in fresh 99% isopropyl alcohol (IPA) in a Anycubic Wash & Cure Plus Machine (Anycubic, Shenzhen, China). An IPA wash in a fresh solution was important to remove any toxic monomers in the uncured resin. Chambers were then removed from the print supports prior to 405nm curing in the same machine, which occurred for 30 minutes on the built-in rotating turntable. Alternatively, we used the 50W 385nm flood lamp (80mW/cm2) (WOWTOU, China) placed face down on a 3D-printed box for 30 minutes, flipping once to avoid shadows.

Chamber assembly

A 30mm diameter, 15cm wide craft roller was used to spread silicone RTV glue (SS-433T, Silicone Solutions, OH, USA) onto a sheet of phenolic resin. Once the glue was spread evenly, the roller was used to transfer it to the bottom surface of the inverted chamber. A 22x22mm #1.5 glass coverslip (VWR, USA) was then pressed onto the glue face with a 3D printed tamper block (S1 Video). The glue was allowed to fully cure for 16hrs in the humid environment of a tissue culture incubator, which accelerated the curing time for RTV silicone. Curing the chamber fully in high humidity was essential for preventing any potential toxicity from the curing agent. A similar protocol was used for the chamber lids.

Chamber sterilization

In a HEPA tissue culture hood, both chambers and lids were bathed in 70% IPA for 10 minutes, followed by sterile water for 10 minutes, then allowed to dry under UV light. They were then placed in small clear polypropylene bags and UV irradiated prior to storage and use. UV sterilization was completed using either a 30mW/Cm2 365nm ELC-500 UV (ELC-500, Electro-Lite, USA) chamber on a rotating platform for 10 minutes per side, or the flood lamp curing chamber previously described for 30 minutes per side (Fig 6).

DNA damage laser stripe assays

RPE1 human hTERT immortalized cells were used. Culturing conditions, 405nm laser irradiation, and microscopy equipment were as described elsewhere [13]. PARP1 enzyme was visualized with an anti-PARP1 chromobody, which comprises the anti-PARP1 VHH derived from alpaca heavy chain antibody genetically fused to TagRFP (Chromotech, Gmbh) to visualize endogenous PARP1.

Cell toxicity and stress assays

TruHD-Q50Q40F human fibroblast cells immortalized with hTERT were cultured as described previously [11] for these experiments. Mitochondrial morphology was assessed in Mod3D and commercial plates using confocal microscopy, mitochondrial Mitotracker and nuclear NucBlue probes (Molecular Probes). Viability was assessed between Mod3D and commercial plates using microscopy of a LIVE/DEAD dye kit (Molecular Probes). Both mitochondrial and viability assays were quantified using CellProfiler (https://cellprofiler.org/), and statistically analyzed using a simple random sample and a Mann-Whitney nonparametric t-test.

Discussion

Early experiments in prototyping these modular designs used low-cost commercial resins, which resulted in cellular toxicity. Presumably, this was due to unpolymerized monomers that did not wash out during the isopropanol washes and subsequently solubilized in media. Thus, wash steps using fresh isopropyl alcohol were essential for this protocol. Within this industry, there are over 40 different resin formulas available. However, the exact formulations are elusive and typically guarded as an industry secret. Most resins are liquid in a methyl ethyl ketone (MEK) base, contributing to a significant odor, with MEK indicated as an irritant. Therefore all printing and handling of liquid resin were done within a chemical fume hood using gloves and goggles for handling. MEK itself is toxic and must be washed away using isopropanol. In combination with the post-printing UV curing step, isopropanol washes coincidently resulted in sterile prints.

UV resins are typically polymers of unsaturated polyester, which is an initiator and a photosensitizer [14]. The initiator is triggered by energy absorbed by the photosensitizer and is used to catalyze the polymerization. Typical polymers sold to hobby consumers are in the methacrylate family and are known to have toxic properties [15, 16]. However, the industry has recently evolved to produce more biocompatible polymers termed “bio inks” [17, 18]. These include polycaprolactone, PLA and polyglycolic acid (PGA) and are FDA-approved for biological use [14, 19, 20]. Of these, PLA resin (e-resin PLA, BioPhotopolymer, eSun, China) was affordable and available in our region. One unanticipated advantage to PLA plastic was the lack of surface charge seen in polystyrene products. Surface charges can lead to liquid transfer problems and have a high meniscus effect in small wells. We did not experiment above 16-wells per 22x22 chamber (or 96-wells per holder), but the limitation of the printer resolution should not be a factor for higher well densities. MSLA resin prints have a higher resistance to temperature than FDM PLA prints, and display higher flexibility with no deformation up to 100°C.

In early 22x50mm designs, we did note the expansion of chamber dimensions by up to 1%. The expansion of the chamber was due to the absorbance of moisture by the plastic within the tissue culture incubator and resulted in a 0.5mm warp on the chamber glass. This is because over three days, the plastic that was bonded to the glass expanded, while the glass coverslip did not. This warping was resolved using either a linked 22x22mm twin chamber design or by using 22x22mm chambers in a 6-chamber holder.

The modular nature of the chambers allows for efficient use of the wells based on experimental need, and therefore there is less waste from unused wells in commercially available multiwell plates. The chamber carriers, the chambers and the lid frames were made from biodegradable PLA resin or filament [21]. The cost savings were dramatic, with a chamber costing under 30 cents, in contrast to some of the commercial examples charging over US$10. One practical aspect is the flexibility to integrate new designs. Prototypes can be generated at minimal cost in just a few hours, without the practical limitations of a local machine shop. MSLA printing time, unlike FDM, does not scale with replicates; printing one, four or eight chambers is completed at the same time. This is because the time-limiting step in MSLA is dictated by the height of the objects in the print area, not the number of objects, as all object layers are simultaneously exposed to UV mask. With FDM, print time is dictated by height, in Z, and tool movement of the extruder head, in XY. Newer MSLA printers use 4X or 8X larger screens (based on consumer computer tablet 4K or 8K LCDs), which could generate 16–32 22x50mm or 32–64 22x22mm chambers in the same 90 minute time-frame. Similarly, inexpensive consumer FDM printers on coreXY design are now available at printing speeds of up to 250mm/sec, or five times the speed of typical printers of a few years ago.

The advantages of additive local manufacturing all come into play with these designs: low cost, minimized inventory maintenance, no lag times between ordering, decreased worrying about availability and shipping costs. The assembled modular nature also means the chamber holder and lids can be reused after sterilization. The craft skills and hands-on time per step required are minimal, and a naive user can generate up to 40 chambers a day (S1 Video).

The cost of 3D printing materials and equipment is now much lower because of the expanding home consumer market. Additionally, we used a black light 50W 385nm LED flood lamp array as an inexpensive post-print processing equipment for curing and sterilization. For total equipment investment, cost recovery is realized in under 100 chambers. The 3D printing designs are freely and openly distributed on a curated website (https://3dprint.nih.gov/) (ID: 3DPX-016293) under Creative Commons license CC-BY-NC-SA. With more submissions from users for unique applications in the future, this repository should grow in designs since the cycle from concept to prototype and the ability to generate experiment-specific designs is now reduced to a negligible time and cost.

Gluing and assembly of Mod3D chambers to glass slides and incubation for curing.

(MP4)

Click here for additional data file.

Extended Live cell DNA damage laser stripe assay data from Fig 5, over 134 seconds, with timestamp.

(MP4)

Click here for additional data file.

(PDF)

Click here for additional data file.

23 Feb 2022

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The paper “Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders” by Gross et al provides a new method for using resin-based 3D printing to produce customized chambers for live-cell microscopy. Unlike previous papers addressing the same issue, this paper found an approach to use resin/MSLA printing to produce these chambers. This offers several advantages over filament printing used by previous studies, including higher print resolution, flatter surfaces for sealing, and faster print times. However, plastic toxicity is an issue, with a major advance of this study being identification of a specific resin and washing process which appears to eliminate these toxicity issues. In addition, a video has been provided showing the assembly process, which should ease adoption of this method by other groups as it illustrates parts of the assembly process which may not be easily communicated in text. While overall the paper is quite strong, I would recommend a few edits to strengthen the claims of non-toxicity.

Major Issues:

1. While the authors extensively describe a lack of toxicity of their chosen adhesive and resin, no direct evidence of this is provided to the reader. I would suggest including data measuring the apoptosis of cells cultured in these chambers. The classical annexin v/propidium iodide assay should be more than sufficient for this. Ideally, multiple time points should eb assessed (e.g. 24, 48 and 72 hours culture).

2. Related to issue #1, the possibility that non-lethal changes to cell behaviour may be occurring is not addressed. A measure of cell morphology, mitochondrial function, or something similar – compared to commercial chambers – would help demonstrate a lack of toxic effects on the cells.

Minor Issue:

1. While the STL files are available at the NIH exchange, no where in the body of the text is this indicated. It would be helpful if the link were provided in the methods.

Reviewer #2: This manuscript describes a protocol to print and use a microscopy chamber dedicated to "replace" disposable commercially available ones.

The chamber consists in a generic frame on which several components can be positioned, depending on the desired chamber number, shape and size.

I strongly support the idea that scientists need such openly distributed tools for making available to any lab, devices that can be expensive.

The device itself seems useful enough to deserve publication, although the manuscript needs to be amended.

I would first recommend a serious review of the existing literature about 3D-printed chambers for microscopy. There is not a single article nor a website (many STL files are freely downloadable in dedicated websites), cited in the introduction. The authors only compare their device to single-use, blister protected, sterile culture dishes dedicated to microscopy imaging.

In the introduction MSLA is mispelled, it is actually maskLESS SLA.

There is some confusion about the resin-based printers. SLA in general refers to the technique where a laser is scanned point-by-point, LCD where the light is spatially filtered with the LCD and finally DLP where the light directly comes from a projector.

In general convention as well, MaskLess SLA refers to the soft-lithography machines that are used for printing microfluidic chips, using microscopy objectives and SU8-type resins. And these machines are about several tens of kilo dollars.

The recent availability of LCD machines has made the resin-based printers affordable, but most of the SLA costs a few thousand dollars, a good DLP can reach several ten thousand dollars as well.

Could the authors screen their manuscript again and provide quantitative values (even a range) when evaluating a parameter? Such as “the resolution not up to the standards” or the “FDA superior”.

Some statements are as well blind and overestimated (unless tested): “can be reused with a sterilization cycle indefinitely”. This is most likely not possible.

And yet, sterilisation with UV in plastic bags should be carefully evaluated, since only the parts directly exposed to light could be decontaminated. Actually, the parts are very complex, and it is unlikely that the overall device could be efficiently decontaminated. Yet, biologists in general favours autoclave for decontamination of devices and parts. I am aware that the plastics and resins are not compliant with such extreme temperatures, and thus, other ways have to be found, but readers should also be warned of the risks.

I have also concerns about the “biology” section. This is indeed necessary to provide a section where the device is in use and to document the features that matter. Especially, it would be useful to provide a more detailed characterisation of the following parameters: low magnification image of a cell mat in the different type of compartments with close-ups; longer time-lapses over several hours and days to demonstrate the lack of drift (the material being softer than most of the sample holders on stages might be susceptible to bend when used in a heated environment).

Overall, I think the device is useful and could benefit to a great readership. Although since the authors would like to publish this device additionally to making it available in an open repository, the writing should be upgraded to meet the standards of research papers, including citing properly the existing literature.

**********

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Reviewer #1: No

Reviewer #2: No

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11 May 2022

Thank you to the reviewers for their comments and insights. Some of these comments were addressed in the detailed protocol in this submission, as this is a protocol variant of a PLOS One submission. The manuscript is meant to be read as manuscript and detailed protocol, as instructed to authors on first submission. There seems to be some concern about the validity of this protocol and what was considered before submission. For context: this was an effort over 3 years, with about 40 prototypes, 17 different adhesives and a dozen different resins. Indeed, the project was stalled for months because we could not find a non-toxic resin until PLA resins appeared in the commercial market. We also spent months on both silicone RTV and UV adhesives from multiple manufacturers, finding toxicity contrary to claims form manufacturers. We also spent months establishing a sterilization protocol that involves both chemical and UV steps, with multiple UV exposures prior to storage and before use, again, outlined in detail in the supplementary protocol.

Reviewer #1: The paper “Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders” by Gross et al provides a new method for using resin-based 3D printing to produce customized chambers for live-cell microscopy. Unlike previous papers addressing the same issue, this paper found an approach to use resin/MSLA printing to produce these chambers. This offers several advantages over filament printing used by previous studies, including higher print resolution, flatter surfaces for sealing, and faster print times. However, plastic toxicity is an issue, with a major advance of this study being identification of a specific resin and washing process which appears to eliminate these toxicity issues. In addition, a video has been provided showing the assembly process, which should ease adoption of this method by other groups as it illustrates parts of the assembly process which may not be easily communicated in text. While overall the paper is quite strong, I would recommend a few edits to strengthen the claims of non-toxicity.

Major Issues:

1. While the authors extensively describe a lack of toxicity of their chosen adhesive and resin, no direct evidence of this is provided to the reader. I would suggest including data measuring the apoptosis of cells cultured in these chambers. The classical annexin v/propidium iodide assay should be more than sufficient for this. Ideally, multiple time points should eb assessed (e.g. 24, 48 and 72 hours culture).

We did provide images of cell growth in the Supplemental Figure 1. We have now moved this into the manuscript in Figure 7A. We now include image data on cell growth, general toxicity by live/dead assay, as apoptosis assays would only assay one type of cell death (7B) and mitochondrial ellipticity with over >80,000 mitochondria assessed (7C).

2. Related to issue #1, the possibility that non-lethal changes to cell behaviour may be occurring is not addressed. A measure of cell morphology, mitochondrial function, or something similar – compared to commercial chambers – would help demonstrate a lack of toxic effects on the cells.

This is now added as Figure 7C and is extremely sensitive given the high n values.

Minor Issue:

1. While the STL files are available at the NIH exchange, no where in the body of the text is this indicated. It would be helpful if the link were provided in the methods.

The URL was provided at the top of the protocol (supplemental protocol) and the ID: 3DPX-016293 number was provided on line 296. We will now clarify the URL in both locations.

Reviewer #2: This manuscript describes a protocol to print and use a microscopy chamber dedicated to "replace" disposable commercially available ones.

The chamber consists in a generic frame on which several components can be positioned, depending on the desired chamber number, shape and size.

I strongly support the idea that scientists need such openly distributed tools for making available to any lab, devices that can be expensive.

The device itself seems useful enough to deserve publication, although the manuscript needs to be amended.

I would first recommend a serious review of the existing literature about 3D-printed chambers for microscopy. There is not a single article nor a website (many STL files are freely downloadable in dedicated websites), cited in the introduction. The authors only compare their device to single-use, blister protected, sterile culture dishes dedicated to microscopy imaging.

In the introduction MSLA is mispelled, it is actually maskLESS SLA.

There is some confusion about the resin-based printers. SLA in general refers to the technique where a laser is scanned point-by-point, LCD where the light is spatially filtered with the LCD and finally DLP where the light directly comes from a projector.

In general convention as well, MaskLess SLA refers to the soft-lithography machines that are used for printing microfluidic chips, using microscopy objectives and SU8-type resins. And these machines are about several tens of kilo dollars.

The recent availability of LCD machines has made the resin-based printers affordable, but most of the SLA costs a few thousand dollars, a good DLP can reach several ten thousand dollars as well.

One point of this protocol is that consumer 3D printers have drastically reduced the costs of printing chambers like these, while depending on specialty resins and more expensive SLA printers would make the costs not worth the effort. Around 2019, low cost resin printers appeared on the market and the consumer printer industry is distinguishing them from DLP or Galvanometer-based printers by referring to them as Masked Stereo Lithography Apparatus or MSLA. This is confusing with the chip industry, which previously used the term Maskless SLA as the reviewer correctly points out. The printer we used here literally casts an LCD generated mask across a UV LED array. In the consumer 3D printing vernacular, MSLA refers to masked SLA. The following are a few typical references to this from industry:

https://www.structo3d.com/pages/mask-stereolithography-msla-technology

https://satori-tech.io/blog/msla-vs-fdm-printing-technique

https://4dfiltration.com/resources/resin-faq/lcd-masked-sla-vs-dlp-resin-printing.html

https://www.oliver3d.com/

Could the authors screen their manuscript again and provide quantitative values (even a range) when evaluating a parameter? Such as “the resolution not up to the standards” or the “FDA superior”.

Some statements are as well blind and overestimated (unless tested): “can be reused with a sterilization cycle indefinitely”. This is most likely not possible.

We had added more info on typical print resolutions for FDM versus MSLA printers, however this was in the introduction:

“A platform controlled by a stepper motor can then provide resolutions in Z as little as 25 micrometers, with an XY resolution below 100 micrometers”

Unsure about the term “FDA superior” as it does not appear in this manuscript.

Some parts are simply holders and can be reused over and over. We have typical stage holder in use for years at this point, with a periodic 70% ethanol bath sterilization, and for stage holder, sterility is not critical. Chambers themselves are one use and disposable.

And yet, sterilisation with UV in plastic bags should be carefully evaluated, since only the parts directly exposed to light could be decontaminated. Actually, the parts are very complex, and it is unlikely that the overall device could be efficiently decontaminated. Yet, biologists in general favours autoclave for decontamination of devices and parts. I am aware that the plastics and resins are not compliant with such extreme temperatures, and thus, other ways have to be found, but readers should also be warned of the risks.

From the detailed protocol, UV is not the sole sterilization step, nor is one step in UV alone used. There is a chemical disinfection step. By experience at this point, at >500 chambers used, we see no more contamination events than commercial gamma irradiated chambers. We will add a warning in the protocol, as the reviewer is correct that biology labs tend to heat sterilize most apparatus.

I have also concerns about the “biology” section. This is indeed necessary to provide a section where the device is in use and to document the features that matter. Especially, it would be useful to provide a more detailed characterisation of the following parameters: low magnification image of a cell mat in the different type of compartments with close-ups; longer time-lapses over several hours and days to demonstrate the lack of drift (the material being softer than most of the sample holders on stages might be susceptible to bend when used in a heated environment).

We did test the chambers for any long term warping within days of 37C incubation. This led to a change in design away from using 22x50mm slides. This was discussed in lines 266 to 271:

“In early 22x50mm designs, we did note the expansion of chamber dimensions by up to 1%. The expansion of the chamber was due to the absorbance of moisture by the plastic within the tissue culture incubator and resulted in a 0.5mm warp on the chamber glass. This is because over three days, the plastic that was bonded to the glass expanded, while the glass coverslip did not. This warping was resolved using either a linked 22x22mm twin chamber design or by using 22x22mm chambers in a 6-chamber holder.”

For stage holders, we used either PLA or PETG, which have glass transition temperatures of 50-80C, far above 37C incubation conditions. With new references added, we should not need to re-establish the stability of these plastics for microscopy.

We have moved cell imaging data from supplemental Figure 1, in addition to the laser stripe experiment in the original version, plus a high data number measure of any mitochondrial stress, as suggested by both reviewers, as well as a general live/dead assay comparing Mod3D to commercial chambers. This is now in Figure 7. Both reviewers raised this point and we agree this is important to validate the utility of the chambers and this justifies addition to the manuscript as Figure 7. Note at even very high data point counts >80,000, we could not see differences in mitochondrial ellipticity, even in these non-transformed cell lines that are very sensitive to stress being derived from Huntington’s disease patients.

Overall, I think the device is useful and could benefit to a great readership. Although since the authors would like to publish this device additionally to making it available in an open repository, the writing should be upgraded to meet the standards of research papers, including citing properly the existing literature.

We had a very difficult time searching the literature for consumable microscopy chambers. We found a chamber system for zebrafish models, and molds for silicone chambers, these references are now added as well as some broader references on the use of 3D printing on open sourced projects. We added 9 additional references to the revised manuscript.

Submitted filename: Response to reviews.docx

Click here for additional data file.

19 May 2022

Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders

PONE-D-22-00858R1

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25 May 2022

PONE-D-22-00858R1

Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders

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