Microfluidic Devices For High Throughput And Long-term Cell Spheroid Culture

In contrast to traditional 2D-monolayer cell cultures,the 3D cell spheroids can better recapitulate the in-vivo features,including cell-cell,cell-ECM,and cell-microenvironment interactions.In-vitro spheroids provide similar metabolism(e.g.,nutrients,oxygen,and metabolites)and proliferation gradients to those found in-vivo,enabling tracking the diffusion path of drugs or metabolites.So,it is an ideal model for cell research.Compared to animal models,3D cell spheroids have the advantages of low cost,short experimental period,scalable preparation,convenient real-time monitoring,and no animal ethical issues.Due to its unique advantages,3D cell spheroid culture has been widely used in cell biology and medical research in recent years.It has become an powerful tool for research in new drug screening,disease modeling,toxicology testing,tissue engineering,and regenerative medicine.In order to promote the widespread application of 3D cell spheroids,researchers have established a large number of cell spheroid culture methods.Although these methods can realize the culture of cell spheroids,these methods have limitations in the easiness of operation,scalability and cost of production,reproducibility and uniformity of spheroids,stability and long-term performance of cell spheroid preparation.To overcome the shortcomings of existing cell spheroid culture methods and promote the popularization and wide application of cell spheroid culture technology,this paper thesis has presented several different cell spheroid culture chips.The main contents list as follows:(1)To overcome the major technical limitations of the traditional hanging drop cell spheroid culture methods,a medium-reservoir-integrated superhydrophobic(MRI-SH)substrate to develop a hanging drop(HD)platform was proposed,which capable of sustaining a long-term culture while maintaining the same advantages as traditional hanging droplet platforms.Due to the extreme wettability contrast between the wettable spots and superhydrophobic surrounding areas,cell suspensions dispensed onto the superhydrophobic patterns can adhere to the wettable spots and form an array of separated quasi-spherical microdroplets.The quasi-spherical shape of hanging drops promotes the agglomeration of cells,and benefits to form tighter cell spheroids with better roundness.Both the experimental results and the simulation analysis confirmed that the chip solved the problem of medium exchange during the long-term cell spheroids culture.It realize the long-term culture(30 days)of cell spheroids without medium exchange.Moreover,87.5% of the cells in the cell spheroids had good viability after 30 days of culture.But the cell spheroids cultured by the traditional hanging drop without medium exchange began necrosis in many area of a spheroid.In addition,due to the smaller touch area of the hanging droplets,the chip cultured more round cell spheroids(roundness was 0.891)than the traditional method.And most of the cell spheroids cultured from the traditional hanging drop were flat(roundness was 0.494).(2)In view of the poor stability of hanging drop in the traditional hanging drop cell spheroid culture method,a novel sitting drop cell spheroid culture chip was developed.The chip contains an array of superhydrophobic microwells that facilitates the self-discretization and self-docking of cell suspensions with the combination of surface tension and gravity,and a thru-hole at the middle of each superhydrophobic microwell bottom that ensures the adequate supply of oxygen to cell aggregations.It can complete the sample loading of 384-well within 3 minutes.This culture mode not only maintains the advantages of low exogenous matrix interference and high atmosphere supply of the hanging drop cell spheroid culture method,but also the cell droplets to be cultured have strong mechanical stability.In addition,its open feature also makes the medium refreshment and cell spheroid retrieval very convenient.Particularly,due to non-wettability of its superhydrophobic surface,it can achieve the rapid collection of cell spheroids in batches(retrieval almost 100% of cell spheroids in 10 minutes).Thus greatly improving the efficiency of cell spheroid production,which was unrealized for other culture methods.(3)To further improve the automation of cell spheroid culturing processe,a curved microwell array cell spheroid culture chip integrated with a automatic medium perfusion unit was developed.The automatic medium perfusion unit employs gravity to drive medium fluid without the need for complex external pumping equipment,which can make the liquid speed in 1 m L/h.In addition,a rapid and economical strategy of machining and caramel reflow process(80℃ reflow in vacuum for 30 minutes)is used for fabricating the curved microwell array chip.By combining the automatic medium perfusion system with the curved microwell array chip,the long-term and dynamic spheroid culture was achieved.Compare to the static culture,98.6% of the cell spheroids cultured from the dynamic culture were live in 3 days and 5.68% of the cell spheroids in the static culture were live(in the sealed chip).Due to the simple,low cost,and without additional mechines drive the liquid to fluid.It is potential to promote the wide application of cell spheroid culture technology in ordinary laboratories.(4)To further enhance the throughput of spheroid production,a self emulsion-based spheroid culture chip was developed.Due to template-guided dewetting effect,this chip spontaneously and rapidly discretizes a cell suspension solution into a large number of droplets,then for ming a large number of cell aggregates in a few minutes and accelerating the cell spheroid culture.After theoretical analysis and calculation,the key geometrical parameters and conditions affecting the self-emulsion efficiency were studied and optimized.By the design of the star-shape with the anti adhesion treatment(by immersing in the Pluronic F-127 solution),an optimal self-emulsion-based microwell array chip for high-throughput cell spheroid culture was fabricated,and the rapid(within in 3 hours)mass production(20000 cell spheroids)of cell spheroids was realized.In summary,this paper proposed four methods to overcome the shortcomings in cell spheroid culture methods at present.The first chip solve the problem of the difficulty of medium exchange during the long-term cell spheroid culture in hanging drop.This chip make it possible to spheroid culture for a month without medium exchange.Then the sessile drop superhydrophobic through hole chip was used to realise the facile operation of cell spheroid culture.It improve the stability of the spheroid culture.After that,concave microwells were fabricated by caramel reflow to perfusion culture with the gravity.Finally,a rapid and high-throughput cell spheroids was prepared with self-emulsification.This paper proposed several cell spheroid culture methods to solve some problems in the traditional spheroid culture.It provide an alternative platform for three-dimensional cell culture such as organoid culture.