我们引进的粒度均匀,并组成控制肿瘤球体的协议大数代(几千到几十万),使用市售微孔板。
肿瘤球状体正日益被视为一个重要的体外模型中对肿瘤细胞在三维空间中的行为。比传统的贴张文化更加生理有关,他们更准确地概括了复杂性和目前的实际肿瘤的相互作用。为了利用这一模型,以更好地评估肿瘤生物学,或新的治疗剂的功效,这是必要的,以便能够重复地产生球状体,以受控的方式并在显著号码。
该AggreWell系统由金字塔形微孔的高密度阵列,成单细胞悬浮液进行离心分离的。细胞的数量聚类在各微孔的底部,并涉及不同的细胞类型的数量和比率仅依靠由实验者引入悬浮液的性质。因此,我们能够生成任意大小和组成的肿瘤与球体出需要修改底层平台技术。反过来数百每培养面积的平方厘米的微孔确保极高的生产水平可以通过一个简单的,非人工密集的过程来实现。因此,我们预计,该协议将是广泛有益的研究人员在肿瘤球体领域。
有越来越多的证据体,肿瘤细胞有不同的行为在三维培养物比它们在塑料培养时以及确定在常规组织培养平台的治疗剂时,转变到多种生理上相关的系统1可能会失去功效。因此,需要研究癌症细胞的行为在这些条件下,既要深入了解其潜在的生物,并且还增加过渡的新的治疗剂的筛选设施到诊所的成功率。一个有用的模型系统,具有悠久的历史采用被称为肿瘤球体1,2癌细胞的三维集群。理想情况下,技术球体的形成将允许生产大量均匀的球体,其大小和组成由实验者所控制的。而悬滴和孔板接近3,4能够满足其中的一些重quirements,吞吐量一般是有限的,并且产生大量的球状物变得劳动密集的任务。
我们最近开发了一个系统,以解决在再生医学领域5类似的挑战。在一系列密集微米级孔的用人被迫聚集( 见图1),该方法允许球状体的生成从任意数量的细胞,包括多种细胞类型6的混合物,以及各种生物材料7的掺入。球状体形成大量-几千到几十万或更多-而可以立即提取或保持在其上,形成具有至少一个8到2(未发表的观察)周培养基交换的微孔。因此,该系统非常适合于大量的均匀和可再现的肿瘤球状体的产生为EFFE的评估ctiveness新型抗肿瘤药物或生物的基本调查。
We have established a system whereby large numbers of uniform spheroids may be generated from multiple cell lines from different sources. We have yet to encounter an adherent cell line that does not form spheroids under these conditions. We have previously observed cell loss in populations prone to anoikis5,8, however to date this issue has not arisen with tumor lines. The system is arbitrarily scalable with surface area, with behavior consistent across microwells in 24-well and 6-well format, as well as prototype bioreactors containing 50,000 microwells each presently under development.
Should spheroid asymmetry be a concern, the incubation time in step 4.8 may be increased to two or three days. Spheroids may also be incubated for a period after extraction from the microwells to increase symmetry, however in this case care must be taken to keep culture densities sufficiently low, as spheroids in contact with one another will often fuse into larger structures. For this reason, we have previously maintained cultures within the microwells in which the aggregates were formed, with the primary limitation being the size of the spheroid. This in turn is a function of both growth rate and initial size8, and if extended culture within the microwell plate is planned, this should be considered in advance. Options to prevent overgrowth, should it occur, include either starting with smaller spheroids, or employing the larger microwells of the AggreWell 800 plate.
Should spheroids fail to increase in coherence over time, one potential cause may be cell death. Particularly in larger aggregates of highly metabolically active cells, mass transport limitations on the delivery of oxygen and essential nutrients can result in a necrotic core2 – thus a non-cohesive spheroid may simply be a consequence of excessive cell death. Alternatively, measurements of the mechanical cohesion of spheroids have been used to assess intercellular binding forces, in relationship to the metastatic potential of a given cell line13. It would be interesting to investigate the relationship between spheroid shape and metastatic potential, perhaps using morphometric parameters such as roundness and perimeter to area ratio.
In addition to investigating the mechanical and morphometric properties of spheroids, assembly in the microwell system permits large-scale production of mixed-composition spheroids, consisting of combinations of multiple cell types and / or biomaterials6,7. The interactions of tumor cells with other cell types are important to more closely model the behavior of tumors in vivo14, thus it may be of interest to generate spheroids from tumor cells in combination with fibroblasts and endothelial cells, for example. Microparticles of various biomaterials may also be incorporated, and can affect spheroid properties both directly through their interactions with cells7,15, and also as reservoirs for the controlled release of growth factors and cytokines into the interior of the spheroid16.
If there is any concern about sterility, for example when working with an microwell plate in which some wells have previously been used, that may have spent some time in an incubator as part of a previous experiment, the wells may be resterilized with 70% ethanol in water.
Once spheroids have been formed, they may also be separated from residual unincorporated cells by passing the suspension over a cell strainer. Individual cells will pass through, while the spheroids will be retained. If the spheroid is suspected of shedding potentially metastatic cells over the course of culture, it may be desirable to perform this procedure multiple times – initially to remove unincorporated cells, and subsequently to isolate purified populations of the cells given off by the spheroids.
The authors have nothing to disclose.
This work was funded by the University of Calgary, under a new investigator start-up grant to Dr. Ungrin.
Name of Reagent/Material | Company | Catalog Number | Comments |
AggreWell 400 plate | StemCell Technologies Inc. | 27845/27945 | |
Rinsing Solution | StemCell Technologies Inc. | 07010 | |
Cell strainer (37 µm) | StemCell Technologies Inc. | 27215 | |
PBS | VWR / LONZA | CA12001-676 | |
Trypsin-Versene (EDTA) | VWR / LONZA | CA12001-660 | |
DMEM | VWR / LONZA | CA12002-212 | |
FBS | VWR / LONZA | CA-95042-112 | |
TrypLE | Invitrogen / Life Technologies | 12605010 | |
Inverted microscope | VWR / Motic | CA19000-610 | |
Allegra X15R centrifuge with carriers for standard well plates | VWR / Beckman | CABKA99465, CABK369704, CABK392806 | |
Laminar flow biosafety cabinet | ESBE / Baker | BKR-SG603AHEUVSP |