Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

Summary

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

Abstract

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 μm microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

Introduction

It is easier to simulate biological in vivo microenvironments with three-dimensional (3D) spheroid cell culture than with two-dimensional (2D) cell culture (e.g., conventional Petri dish cell culture) to produce more physiologically realistic experimental results¹. Currently available spheroid formation methods include the hanging drop technique², liquid-overlay technique³, carboxymethyl cellulose technique⁴, magnetic force-based microfluidic technique⁵, and the use of bioreactors⁶. Although each method has its own benefits, further improvement in reproducibility, productivity, and generating coculture spheroids is necessary. For example, while the magnetic force-based microfluidic technique⁵ is relatively inexpensive, the effects of strong magnetic fields on living cells must be carefully considered. The benefits of spheroid culture, particularly in the study of mesenchymal stem cell differentiation and proliferation, have been reported in several studies^7,8,9.

The centrifugal microfluidic system, also known as lab-on-a-CD (compact disc), is useful for easily controlling the fluid inside and exploiting the rotation of the substrate and has thus been utilized in biomedical applications such as immunoassays¹⁰, colorimetric assays for detecting biochemical markers¹¹, nucleic acid amplification (PCR) assays, automated blood analysis systems¹², and all-in-one centrifugal microfluidic devices¹³. The driving force controlling the fluid is the centripetal force created by rotation. Additionally, multiple functions of mixing, valving, and sample splitting can be done simply in this single CD platform. However, compared to the above-mentioned biochemical analysis methods, there have been fewer trials applying CD platforms to culture cells, especially spheroids¹⁴.

In this study, we show the performance of the centrifugal microfluidic-based spheroid (CMS) system by monoculture or coculture of human adipose-derived stem cells (hASC) and human lung fibroblasts (MRC-5). This paper describes in detail our group's research methodology¹⁵. Thus, the spheroid culture lab-on-a-CD platform can be easily reproduced. A CMS generating system comprising a CMS culture chip, a chip holder, a DC motor, a motor mount, and a rotating platform, is presented. The motor mount is 3D printed with acrylonitrile butadiene styrene (ABS). The chip holder and rotating platform are CNC (computer numerical control) machined with the PC (polycarbonate). The rotational speed of the motor is controlled from 200 to 4,500 rpm by encoding a PID (proportional-integral-derivative) algorithm based on pulse-width modulation. Its dimensions are 100 mm x 100 mm x 150 mm and it weighs 860 g, making it easy to handle. Using the CMS system, spheroids can be generated under various gravity conditions from 1 x g to 521 x g, so the study of cell differentiation promotion under high gravity can be extended from 2D cells^16,17 to 3D spheroid. Coculture of various types of cells is also a key technology for effectively mimicking the in vivo environment¹⁸. The CMS system can easily generate monoculture spheroids, as well as coculture spheroids of various structure types (e.g., concentric, Janus, and sandwich). The CMS system can be utilized not only in simple spheroid studies but also in 3D organoid studies, to consider human organ structures.

Protocol

1. Centrifugal microfluidic-based spheroid (CMS) culture chip fabrication

1. Make PC molds for the top and bottom layers of the CMS culture chip by CNC machining. Detailed dimensions of the chip are given in Figure 1.
2. Mix PDMS base and PDMS curing agent at a ratio of 10:1 (w/w) for 5 min and place in a desiccator for 1 h to remove air bubbles.
3. After pouring the PDMS mixture into the molds of the CMS culture chip, remove air bubbles for 1 h more and cure in a heat chamber at 80 °C for 2 h.
4. Place them in the vacuumed plasma cleaner with the surfaces to be bonded facing up and expose them to air-assisted plasma at a power of 18 W for 30 s.
5. Bond the two layers of the CMS culture chip and place it in the heat chamber at 80 °C for 30 min to increase adhesion strength.
6. Sterilize the CMS culture chip in an autoclave sterilizer at 121 °C and 15 psi.

2. Cell preparation

1. Thaw the 1 mL of the vial containing 5 × 10⁵–1 × 10⁶ hASCs or MRC-5s cells in a water bath at 36.5 °C for 2 min.
2. Add 1 mL of Dulbecco’s Modified Eagle Medium (DMEM) to a vial and gently mix with a 1,000 μL pipette.
3. Put 15 mL of the DMEM prewarmed to 36.5 °C into a 150 mm diameter Petri dish using a pipette and add the cells from the vial.
4. After 1 day, aspirate the DMEM and replace with 15 mL of fresh DMEM. Subsequently, change the media every 2 or 3 days.
5. To detach the cells from the Petri dish, add 4 mL of trypsin to the Petri dishes and place them in an incubator at 36.5 °C and 5% CO₂ for 4 min.

3. Monoculture spheroid formation

1. Put 2.5 mL of 4% (w/v) pluronic F-127 solution into the inlet hole of the CMS culture chip (Figure 2A) while rotating the chip at 500–1,000 rpm and then rotate the chip at 4,000 rpm for 3 min using the CMS system (Figure 2B).
   NOTE:  The pluronic coating prevents cell attachment to the inlet port while the chip rotates. Make sure air is not trapped in the microwells.
2. Incubate the CMS culture chip filled with pluronic solution overnight at 36.5 °C in 5% CO₂.
3. Remove the pluronic solution, wash out the remaining pluronic solution with DMEM, and dry the chip for 12 h on a clean bench.
4. Add 2.5 mL of DMEM to the CMS culture chip and rotate the chip at ~4,000 rpm for 3 min for prewetting the inside of the chip.
5. Stop the rotation and pull out 100 μL of DMEM to make room for injecting the cell suspension.
6. Add 100 μL of cell suspensions that contain either 5 × 10⁵ hASCs or 8× 10⁵ MRC-5s by pipetting Uniformly distribute the cells by pipetting 3–5x for resuspension.
7. Rotate the chip at 3,000 rpm for 3 min to trap cells in each microwell by centrifugal force.
   NOTE:  Excessive rotational speed can cause cell escape through solution ejection holes.
8. Culture the cells for 3 days in the incubator at 36.5 °C, >95% humidity, and 5% CO₂, rotating at 1,000–2,000 rpm. Change culture medium every day.

4. Coculture spheroid formation

1. Concentric spheroids formation
   1. Add the first cells, 2.5 × 10⁵ hASCs, and rotate the chip at 3,000 rpm. After 3 min, add the second cells, 4 × 10⁵ MRC-5s, and rotate the chip at 3,000 rpm for 3 min. Inject a total of 100 μL of cell suspensions by pipetting. When the cells are injected, shift the rotational speed to 500-1,000 rpm.
   2. Culture the cells in the incubator at 36.5 °C, >95% humidity%, and 5% CO₂ by rotating at 1,000–2,000 rpm. The concentric spheroids are created within 24 h. For long-term culture, change the culture medium every day.
2. Janus spheroid formation
   1. Add 100 μL of cell suspensions containing the first cells, 2.5 × 10⁵ hASCs, by pipetting while the chip rotates at 500–1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.
   2. Incubate the chip at 36.5 °C, >95% humidity, and 5% CO₂ by rotating at 1,000–2,000 rpm for 3 h.
   3. Add 100 μL of the cell suspensions containing the second set of cells, 4 × 10⁵ MRC-5s, by pipetting while the chip rotates at 500–1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.
   4. Culture the cells in the incubator at 36.5 °C, >95% humidity, and 5% CO₂ by rotating at 1,000–2,000 rpm. The Janus spheroids are created within 24 h. For long-term culture, change the culture medium every day.
3. Sandwich spheroid formation
   1. Add 100 μL of cell suspensions containing the first cells, 1.5 × 10⁵ hASCs, by pipetting while the chip rotates at 500–1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.
   2. Incubate the chip at 36.5 °C, >95% humidity, and 5% CO₂ by rotating at 1,000–2,000 rpm for 3 h.
   3. Add 100 μL of cell suspensions containing the second cells, 3 × 10⁵ MRC-5s, by pipetting while the chip rotates at 500–1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.
   4. Incubate the chip at 36.5 °C, >95% humidity%, and 5% CO₂ by rotating at 1,000–2,000 rpm for 3 h.
   5. Add 100 μL of cell suspensions containing the third cells, 1.5 × 10⁵ hASCs, by pipetting while the chip rotates at 500–1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.
   6. Culture the cells in the incubator at 36.5 °C, >95% humidity%, and 5% CO₂ by rotating at 1,000–2,000 rpm. The sandwich spheroids are created within 12 h. For long-term culture, change the culture medium every day.

5. Cell staining

1. Warm the cell fluorescence dye to room temperature (20 °C).
2. Add 20 μL of anhydrous dimethylsulfoxide (DMSO) per vial to make a 1 mM solution.
3. Dilute the fluorescence to a final working concentration of 1 μM using DMEM.
4. Add the fluorescence to the cell suspension and gently resuspend using a pipette.
5. Incubate 20 min at 36.5 °C, humidity of >95%, and 5% CO₂.

Results

The 6 cm diameter CMS culture chip (Figure 2) was successfully made following the above protocol. First, the chip was made separately from a top layer and a bottom layer and then bonded together by plasma bonding. Resulting spheroids can be easily gathered by detaching the chip. The channel of the CMS culture chip comprises an inlet port and central, slide, and microwell regions (Figure 3). The cell, medium, and pluronic solutions are injected through an inlet h...

Discussion

The CMS is a closed system in which all injected cells enter the microwell without waste, making it more efficient and economical than conventional microwell-based spheroid generation methods. In the CMS system, the media is replaced every 12–24 h through a suction hole designed to remove the media in the chip (Figure 3A). During the media suction process, barely any media escapes from inside the microwell due to the surface tension between the media and the wall of the microwell. A us...

Materials

Name	Company	Catalog Number	Comments
3D printer	Cubicon	3DP-210F
Adipose-derived mesenchymal stem cells (hASC)	ATCC	PCS-500-011
Antibiotic-Antimycotic	Gibco	15240-062	Contained 1% of completed medium and buffer
CellTracker Green CMFDA	Thermo Fisher Scientific	C2925	10 mM
CellTracker Red CMTPX	Thermo Fisher Scientific	C34552	10 mM
Computer numerical control (CNC) rotary engraver	Roland DGA	EGX-350
DC motor	Nurielectricity Inc.	MB-4385E
Dimethylsulfoxide (DMSO)	Sigma Aldrich	D2650
Dulbecco's modified eaggle's medium (DMEM)	ATCC	30-2002
Dulbecco's phosphate buffered saline (D-PBS)	ATCC	30-2200
Fetal bovine serum	ATCC	30-2020	Contained 10% of completed medium
human lung fibroblasts (MRC-5)	ATCC	CCL-171
Inventor 2019	Autodesk		3D computer-aided design program
Petri dish Φ 150 mm	JetBiofill	CAD010150	Surface Treated
Plasma cleaner	Harrick Plasma	PDC-32G
Pluronic F-127	Sigma Aldrich	11/6/9003	Dilute with phosphate buffered saline to 4% (w/v) solution
Polycarbonate (PC)	Acrylmall	AC15PC	200 x 200 x 15 mm
Polydimethylsiloxane (PDMS)	Dowcorning	Sylgard 184
Trypsin	Gibco	12604021