bioink preparation, acoustic device assembly, and viability analysis of cell spheroids

3D Akustik Montaj Cihazı Kullanarak Tek Tip Hücre Sferoidlerinin Ölçeklenebilir Üretimi

3D in vitro culture models, which provide more in vivo-like structural and morphological characteristics compared to conventional 2D culture models, have been recognized as promising systems in various biomedical applications such as tissue engineering, disease modeling, and drug screening1,2,3. As one type of 3D culture model, cell spheroids typically refer to cell aggregation, creating 3D spheroidal structures characterized by enhanced cell-cell and cell-matrix interactions4,5,6. Therefore, fabricating cell spheroids has become a powerful tool for enabling diverse biological studies.
Various techniques, including hanging drop7, non-adhesive plates8, or microwell devices9, have been developed to obtain spheroids. In principle, these methods commonly facilitate cell assembly by utilizing physical forces such as gravitational force while minimizing interactions between cells and the substrate. However, they often involve labor-intensive processes, have low productivity, and pose challenges for controlling spheroid size10,11. Importantly, the production of spheroids with the desired size and uniformity in sufficient quantity is of utmost importance to satisfy specific biological applications. In contrast to the above-mentioned methods, acoustic waves, as one type of external-force-driven technique12,13,14, have shown potential for mass manufacturing of cell spheroids with high quality and throughput, based on the principle of enhancing cell aggregation through external forces15,16,17,18. Unlike electromagnetic or magnetic forces, acoustic-based cell manipulation techniques are non-invasive and label-free, enabling spheroid formation with excellent biocompatibility19,20.
Commonly, standing surface acoustic waves (SAWs) and bulk acoustic waves (BAWs)-based devices have been developed to generate spheroids, utilizing the acoustic nodes (ANs) produced by corresponding standing acoustic fields21,22,23. Particularly, acoustic assembly devices based on BAWs, with the merits of convenient manufacture, easy operation, and excellent scalability, have gained attention for fabricating cell spheroids24,25. We have recently developed a facile BAWs-based acoustic assembly device with the ability to generate spheroids with high throughput26. The proposed device consists of a square polymethyl methacrylate (PMMA) chamber with three lead zirconate titanate (PZT) transducers arranged respectively in the X/Y/Z plane. This arrangement enables the creation of a 3D dot-array pattern of levitated acoustic nodes (LANs) for driving cell assembly. Compared to previously reported BAWs- or SAWs-based devices, which can only create a 1D or 2D array of ANs27,28,29, the present device enables a 3D dot-array of LANs for rapid cell aggregate formation within the gelatin methacryloyl (GelMA) solution. Subsequently, cell aggregates matured into spheroids with high viability within the photocured GelMA scaffolds after three days of cultivation. Finally, a large number of spheroids with uniform size were easily obtained from the GelMA scaffolds for downstream applications.

Yazar Spotlight: Yüksek Kaliteli 3D Hücre Küresel Kültürü için İskelesiz Akustik Montaj Yönteminin Geliştirilmesi 

Hücre sferoidlerinin seri üretimi için üç boyutlu akustik montaj cihazı

3D in vitro culture models, like cell spheroids, have been widely used in the fields of tissue engineering, disease modeling, and drug screening. Our research focuses on developing and invest per engineering method to fabricate spheroids or other 3D in vitro culture models with high quality for diverse biological studies. In the current protocol, the spheroids grew within the GelMA scaffold and are retrieved at the final layback, dissolving the scaffold, causing additional procedures.We will try to acoustically assemble cell aggregates in a cultural medium to enable them to grow within spheroids without scaffold. Compared to magnetic assembly techniques, acoustic waves can directly assemble cells in a label-free manner without potential toxic effects on cells. Besides, our protocol enables cell spheroids to be assembled in a three-dimensional array, substantially increasing throughput.

Watch this Scientific Journal Video about Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids at JoVE.com

Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids  

Biyomürekkep Hazırlama, Akustik Cihaz Montajı ve Hücre Sferoidlerinin Canlılık Analizi

Biyo mürekkep solüsyonunu hazırlamak için uygun miktarda C3A hücre süspansiyonunu sterilize edilmiş jelMA solüsyonu ile karıştırın. C3A hücre sferoidlerini iki mikromolar hücre izleyici solüsyonu ile 37 derecede 20 dakika önceden boyayın. Hücreleri yıkamak için önce süpernatini santrifüjleyerek çıkarın ve ardından taze hücre kültürü ortamı ekleyin.Sterilize edilmiş akustik cihaz haznesine en az iki mililitre biyo mürekkep ekleyin. Bir 3D hücre agregası dizisi oluşturmak üzere her bir PZT dönüştürücünün çalıştırılmasını başlatmak için işlev üretecini ve güç amplifikatörünü açın. 3D hidrojel iskeleler oluşturmak için 30 saniye boyunca mavi ışık kullanarak biyo mürekkebi çapraz bağlayın ve birleştirilmiş hücre agregalarını akustik olarak kapsülleyin.Ardından, 3D hidrojel iskelesini hazneden dikkatlice bir Petri kabına aktarın. Temiz bir tıraş bıçağı kullanarak küçük parçalara ayırın ve ardından Petri kabına hücre kültürü ortamı ekleyin. C3A hücre sferoidlerini, bir mikrolitre kalsiyum ve iki mikrolitre propidyum iyodür içeren bir mililitre PBS'de 37 santigrat derecede 15 dakika boyunca farklı kültür zaman noktalarında inkübe edin.Hidrojel iskelelere gömülü numuneyi PBS ile iki kez durulayın. Alınan sferoidler için, beş dakika boyunca 200 G'de santrifüjleme yapın ve PBS ile iki kez yıkayın. Bir floresan mikroskobu kullanarak küreleri gözlemleyin ve görüntüleri yakalayın.Hücre agregaları, yeşil bir floresan sinyali ile normal 3D nokta dizisi modelinde düzenlendi. Birleştirilen C3A gelMA agregaları, sferoid çapında bir artışla birlikte üçüncü günde kademeli olarak entegre edildi ve sıkı sferoidler oluşturdu. Hücre sferoidlerinin canlılığı üçüncü günden önce iyi kaldı, ancak bir haftalık kültürden sonra biraz azaldı.Serbest bırakılan sferoidler, arzu edilen canlılık ve albümin ekspresyonu ile birlikte dar bir boyut dağılımı ile iyi bir küresel morfolojiyi korudu.

bioink-preparation-acoustic-device-assembly-viability-analysis-cell

Research

JoVE Journal

Bioengineering

Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids

87 Görüntüleme.  Hangzhou Dianzi University. Biyo mürekkep solüsyonunu hazırlamak için uygun miktarda C3A hücre süspansiyonunu sterilize edilmiş jelMA solüsyonu ile karıştırın. C3A hücre sferoidlerini iki mikromolar hücre izleyici solüsyonu ile 37 derecede 20 dakika önceden boyayın. Hücreleri yıkamak için önce süpernatini santrifüjleyerek çıkarın ve ardından taze hücre kültürü ortamı ekleyin.Sterilize edilmiş akustik cihaz haznesine en az iki mililitre biyo mürekkep ekleyin. Bir 3D hücre agregas...

Video: Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids  

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

Biyomühendislik

Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids

To begin, sterilize the chamber of the acoustic device with 75%alcohol for five minutes. Then, clean the device with sterile PBS and irradiate it with UV light for one hour. Mount the sterile acoustic device onto a microscope stage for top-view observation of the chamber's interior.Position a digital microscope on the side of the device that is free from PZT transducers, enabling side-view observation of the chamber's interior. Independently connect the wires from the three PZT transducers in series to three power amplifiers and three output channels of function generators. Program the settings on the function generators for each PCT transducer, specifying parameters such as sinusoidal waveforms, frequency, and amplitude.Culture 3CA cells in DMEM supplemented with 10%FBS and 1%penicillin streptomycin in a T25 cell culture flask. When the cells become 80%confluent, wash the culture twice with PBS. After removing the PBS, add two milliliters of 0.05%trypsin-EDTA to the flask and incubate at 37 degrees Celsius for cell detachment.Add two milliliters of complete cell culture medium into the flask to stop the trypsinization. Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 G for five minutes.

Tek tip hücre sferoidleri üretmek için bir akustik montaj sistemi ve hücre kültürü hasadı kurulması

To prepare the bio ink solution mix an appropriate amount of C3A cell suspension with the sterilized gelMA solution. Prestain the C3A cell spheroids with two micromolar cell tracker solution at 37 degrees for 20 minutes. To wash the cells, first, remove the supernatin by centrifuging and then add fresh cell culture medium.Add at least two milliliters of bio ink into the sterilized acoustic device chamber. Turn on the function generator and power amplifier to initiate the actuation of each PZT transducer to generate a 3D array of cell aggregates. Crosslink the bio ink using blue light for 30 seconds to create 3D hydrogel scaffolds, encapsulating the assembled cell aggregates acoustically.Next, carefully transfer the 3D hydrogel scaffold from the chamber into a Petri dish. Cut it into small pieces using a clean razor, and then add cell culture medium into the Petri dish. Incubate the C3A cell spheroids at different culture time points in one milliliter of PBS containing one microliter of calcium am and two microliters of propidium iodide for 15 minutes at 37 degrees Celsius.Rinse the sample embedded in hydrogel scaffolds twice with PBS. For retrieved spheroids, perform centrifugation at 200 G for five minutes and wash twice with PBS. Using a fluorescence microscope, observe the spheroids and capture the images.The cell aggregates were arranged in the regular 3D dot array pattern with a green fluorescent signal. The assembled C3A gelMA aggregates gradually integrated and formed tight spheroids by day three, accompanied by an increase in spheroid diameter. The viability of the cell spheroids remained good before day three, but slightly decreased after a week of culture.The released spheroids maintained a good spherical morphology with a narrow size distribution along with desirable viability and expression of albumin.