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词条 Microfluidic cell culture
释义

  1. Fabrication

  2. Advantages

  3. Culture Platforms

      Two-dimensional culture    Three-dimensional culture  

  4. References

Microfluidic cell culture integrates knowledge from biology, biochemistry, engineering, and physics to develop devices and techniques for culturing, maintaining, analyzing, and experimenting with cells at the microscale.[1][2] It merges microfluidics, a set of technologies used for the manipulation of small fluid volumes (μL, nL, pL) within artificially fabricated microsystems, and cell culture, which involves the maintenance and growth of cells in a controlled laboratory environment.[3][4] Microfluidics has been used for cell biology studies as the dimensions of the microfluidic channels are well suited for the physical scale of cells.[2] For example, eukaryotic cells have linear dimensions between 10-100 μm which falls within the range of microfluidic dimensions.[4] A key component of microfluidic cell culture is being able to mimic the cell microenvironment which includes soluble factors that regulate cell structure, function, behavior, and growth.[2] Another important component for the devices is the ability to produce stable gradients that are present in vivo as these gradients play a significant role in understanding chemotactic, durotactic, and haptotactic effects on cells.[2]

Fabrication

Some considerations for microfluidic devices relating to cell culture include:

  • fabrication material (e.g., polydimethylsiloxane (PDMS), polystyrene)
  • culture region geometry
  • control system for delivering and removing media when needed using either passive methods (e.g., gravity-driven flow, capillary pumps, or Laplace pressure based ‘passive pumping’) or a flow-rate controlled device (i.e., perfusion system)[5][6][7][1][8]

Fabrication material is crucial as not all polymers are biocompatible, with some materials such as PDMS causing undesirable adsorption or absorption of small molecules.[9][10] Additionally, uncured PDMS oligomers can leach into the cell culture media, which can harm the microenvironment.[9] As an alternative to commonly used PDMS, there have been advances in the use of thermoplastics (e.g., polystyrene) as a replacement material.[11][12]

Spatial organization of cells in microscale devices largely depends on the culture region geometry for cells to perform functions in vivo.[13][14] For example, long, narrow channels may be desired to culture neurons.[13] The perfusion system chosen might also affect the geometry chosen. For example, in a system that incorporates syringe pumps, channels for perfusion inlet, perfusion outlet, waste, and cell loading would need to be added for the cell culture maintenance.[15] Perfusion in microfluidic cell culture is important to enable long culture periods on-chip and cell differentiation.[16]

Other critical aspects for controlling the microenvironment include: cell seeding density, reduction of air bubbles as they can rupture cell membranes, evaporation of media due to an insufficiently humid environment, and cell culture maintenance (i.e. regular, timely media changes).[17][16][18]

Advantages

Some of the major advantages of microfluidic cell culture include reduced sample volumes (especially important when using primary cells, which are often limited) and the flexibility to customize and study multiple microenvironments within the same device.[19] A reduced cell population can also be used in a microscale system (e.g., a few hundred cells) in comparison to macroscale culture systems (which often require 105 – 107 cells); this can make studying certain cell-cell interactions more accessible.[20] These reduced cell numbers make studying non-dividing or slow dividing cells (e.g., stem cells) easier than traditional culture methods (e.g., flasks, petri dishes, or well plates) due to the smaller sample volumes.[20][21] Given the small dimensions in microfluidics, laminar flow can be achieved, allowing manipulations with the culture system to be done easily without affecting other culture chambers.[21] Laminar flow is also useful as is it mimics in vivo fluid dynamics more accurately, often making microscale culture more relevant than traditional culture methods.[22] Compartmentalized microfluidic cultures have also been combined with live cell calcium imaging, where depolarizing stimuli have been delivered to the peripheral terminals of neurons, and calcium responses recorded in the cell body. [23] This technique has demonstrated a stark difference in the sensitivity of the peripheral terminals compared to the neuronal cell body to certain stimuli such as protons. [23] This gives an excellent example as to why it is so important to study the peripheral terminals in isolation using microfluidic cell culture devices.

Culture Platforms

Two-dimensional culture

Two-dimensional (2D) cell culture is cell culture that takes place on a flat surface, e.g. the bottom of a well-plate, and is known as the conventional method.[24] While these platforms are useful for growing and passaging cells to be used in subsequent experiments, they are not ideal environments to monitor cell responses to stimuli as cells cannot freely move or perform functions as observed in vivo that are dependent on cell-extracellular matrix material interactions.[24]

Three-dimensional culture

Three-dimensional (3D) cell culture is cell culture that takes place in a biologically relevant matrix, usually this involves cells being embedded in a hydrogel containing extracellular molecules (e.g., collagen).[24] By adding an additional dimension, more advanced cell architectures can be achieved, and cell behavior is more representative of in vivo dynamics; cells can engage in enhanced communication with neighboring cells and cell-extracellular matrix interactions can be modeled.[24][25] These simplified 3D cell culture models can be combined in a manner that recapitulates tissue- and organ-level functions in devices known as organ-on-a-chip.[24] In these devices, chambers or collagen layers containing different cell types can interact with one another for multiple days while various channels deliver nutrients to the cells.[24][26] An advantage of these devices is that tissue function can be characterized and observed under controlled conditions (e.g., effect of shear stress on cells, effect of cyclic strain or other forces) to better understand the overall function of the organ.[24][27] While these 3D models ofter better model organ function on a cellular level compared with 2D models, there are still challenges. Some of the challenges include: imaging of the cells, control of gradients in static models (i.e., without a perfusion system), and difficulty recreating vasculature.[27] Despite these challenges, 3D models are still used as tools for studying and testing drug responses in pharmacological studies.[24] In recent years, there are microfluidic devices reproducing the complex in vivo microvascular network. The device is able to create a physiologically realistic 3D environment, which is desirable as a tool for drug screening, drug delivery, cell-cell interactions, tumor metastasis etc.[28][29]

References

1. ^1 {{cite journal | vauthors = Bhatia SN, Ingber DE | title = Microfluidic organs-on-chips | language = En | journal = Nature Biotechnology | volume = 32 | issue = 8 | pages = 760–72 | date = August 2014 | pmid = 25093883 | doi = 10.1038/nbt.2989 }}
2. ^1 2 3 {{cite journal | vauthors = Young EW, Beebe DJ | title = Fundamentals of microfluidic cell culture in controlled microenvironments | journal = Chemical Society Reviews | volume = 39 | issue = 3 | pages = 1036–48 | date = March 2010 | pmid = 20179823 | pmc = 2967183 | doi = 10.1039/b909900j }}
3. ^{{cite journal | vauthors = Mehling M, Tay S | title = Microfluidic cell culture | journal = Current Opinion in Biotechnology | volume = 25 | pages = 95–102 | date = February 2014 | pmid = 24484886 | doi = 10.1016/j.copbio.2013.10.005 }}
4. ^1 {{cite journal | vauthors = Whitesides GM | title = The origins and the future of microfluidics | language = En | journal = Nature | volume = 442 | issue = 7101 | pages = 368–73 | date = July 2006 | pmid = 16871203 | doi = 10.1038/nature05058 | bibcode = 2006Natur.442..368W }}
5. ^{{Cite journal|last=Cho|first=Brenda S.|last2=Schuster|first2=Timothy G.|last3=Zhu|first3=Xiaoyue|last4=Chang|first4=David|last5=Smith|first5=Gary D.|last6=Takayama|first6=Shuichi|date=2003-04-01|title=Passively Driven Integrated Microfluidic System for Separation of Motile Sperm|journal=Analytical Chemistry|volume=75|issue=7|pages=1671–1675|doi=10.1021/ac020579e|issn=0003-2700}}
6. ^{{cite journal | vauthors = Zimmermann M, Schmid H, Hunziker P, Delamarche E | title = Capillary pumps for autonomous capillary systems | journal = Lab on a Chip | volume = 7 | issue = 1 | pages = 119–25 | date = January 2007 | pmid = 17180214 | doi = 10.1039/b609813d }}
7. ^{{cite journal | vauthors = Walker G, Beebe DJ | title = A passive pumping method for microfluidic devices | journal = Lab on a Chip | volume = 2 | issue = 3 | pages = 131–4 | date = August 2002 | pmid = 15100822 | doi = 10.1039/b204381e | citeseerx = 10.1.1.118.5648 }}
8. ^{{cite journal | vauthors = Kim L, Toh YC, Voldman J, Yu H | title = A practical guide to microfluidic perfusion culture of adherent mammalian cells | journal = Lab on a Chip | volume = 7 | issue = 6 | pages = 681–94 | date = June 2007 | pmid = 17538709 | doi = 10.1039/b704602b }}
9. ^1 {{cite journal | vauthors = Regehr KJ, Domenech M, Koepsel JT, Carver KC, Ellison-Zelski SJ, Murphy WL, Schuler LA, Alarid ET, Beebe DJ | title = Biological implications of polydimethylsiloxane-based microfluidic cell culture | journal = Lab on a Chip | volume = 9 | issue = 15 | pages = 2132–9 | date = August 2009 | pmid = 19606288 | pmc = 2792742 | doi = 10.1039/b903043c }}
10. ^{{cite journal | vauthors = Halldorsson S, Lucumi E, Gómez-Sjöberg R, Fleming RM | title = Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices | journal = Biosensors & Bioelectronics | volume = 63 | pages = 218–231 | date = January 2015 | pmid = 25105943 | doi = 10.1016/j.bios.2014.07.029 }}
11. ^{{cite journal | vauthors = Berthier E, Young EW, Beebe D | title = Engineers are from PDMS-land, Biologists are from Polystyrenia | journal = Lab on a Chip | volume = 12 | issue = 7 | pages = 1224–37 | date = April 2012 | pmid = 22318426 | doi = 10.1039/c2lc20982a }}
12. ^{{cite journal | vauthors = van Midwoud PM, Janse A, Merema MT, Groothuis GM, Verpoorte E | title = Comparison of biocompatibility and adsorption properties of different plastics for advanced microfluidic cell and tissue culture models | journal = Analytical Chemistry | volume = 84 | issue = 9 | pages = 3938–44 | date = May 2012 | pmid = 22444457 | doi = 10.1021/ac300771z }}
13. ^1 {{cite journal | vauthors = Rhee SW, Taylor AM, Tu CH, Cribbs DH, Cotman CW, Jeon NL | title = Patterned cell culture inside microfluidic devices | journal = Lab on a Chip | volume = 5 | issue = 1 | pages = 102–7 | date = January 2005 | pmid = 15616747 | doi = 10.1039/b403091e | hdl = 10371/7982 }}
14. ^{{cite journal | vauthors = Folch A, Toner M | title = Cellular micropatterns on biocompatible materials | journal = Biotechnology Progress | volume = 14 | issue = 3 | pages = 388–92 | date = 1998-01-01 | pmid = 9622519 | doi = 10.1021/bp980037b }}
15. ^{{cite journal | vauthors = Hung PJ, Lee PJ, Sabounchi P, Lin R, Lee LP | title = Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays | journal = Biotechnology and Bioengineering | volume = 89 | issue = 1 | pages = 1–8 | date = January 2005 | pmid = 15580587 | doi = 10.1002/bit.20289 }}
16. ^1 {{cite journal | vauthors = Tourovskaia A, Figueroa-Masot X, Folch A | title = Differentiation-on-a-chip: a microfluidic platform for long-term cell culture studies | journal = Lab on a Chip | volume = 5 | issue = 1 | pages = 14–9 | date = January 2005 | pmid = 15616734 | doi = 10.1039/b405719h }}
17. ^{{cite journal | vauthors = Meyvantsson I, Beebe DJ | title = Cell culture models in microfluidic systems | journal = Annual Review of Analytical Chemistry | volume = 1 | issue = 1 | pages = 423–49 | date = 2008-06-13 | pmid = 20636085 | doi = 10.1146/annurev.anchem.1.031207.113042 | bibcode = 2008ARAC....1..423M }}
18. ^{{cite journal | vauthors = Yu H, Alexander CM, Beebe DJ | title = Understanding microchannel culture: parameters involved in soluble factor signaling | journal = Lab on a Chip | volume = 7 | issue = 6 | pages = 726–30 | date = June 2007 | pmid = 17538714 | doi = 10.1039/b618793e }}
19. ^{{cite journal | vauthors = Mehling M, Tay S | title = Microfluidic cell culture | journal = Current Opinion in Biotechnology | volume = 25 | pages = 95–102 | date = February 2014 | pmid = 24484886 | doi = 10.1016/j.copbio.2013.10.005 }}
20. ^1 {{cite journal | vauthors = Halldorsson S, Lucumi E, Gómez-Sjöberg R, Fleming RM | title = Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices | journal = Biosensors & Bioelectronics | volume = 63 | pages = 218–231 | date = January 2015 | pmid = 25105943 | doi = 10.1016/j.bios.2014.07.029 }}
21. ^1 {{cite journal | vauthors = Gómez-Sjöberg R, Leyrat AA, Pirone DM, Chen CS, Quake SR | title = Versatile, fully automated, microfluidic cell culture system | journal = Analytical Chemistry | volume = 79 | issue = 22 | pages = 8557–63 | date = November 2007 | pmid = 17953452 | doi = 10.1021/ac071311w }}
22. ^{{cite journal | vauthors = Cimetta E, Vunjak-Novakovic G | title = Microscale technologies for regulating human stem cell differentiation | journal = Experimental Biology and Medicine | volume = 239 | issue = 9 | pages = 1255–63 | date = September 2014 | pmid = 24737735 | pmc = 4476254 | doi = 10.1177/1535370214530369 }}
23. ^1 {{Cite journal|last=Clark|first=Alex J.|last2=Menendez|first2=Guillermo|last3=AlQatari|first3=Mona|last4=Patel|first4=Niral|last5=Arstad|first5=Erik|last6=Schiavo|first6=Giampietro|last7=Koltzenburg|first7=Martin|date=2018|title=Functional imaging in microfluidic chambers reveals sensory neuron sensitivity is differentially regulated between neuronal regions:|url=http://insights.ovid.com/crossref?an=00006396-201807000-00023|journal=PAIN|language=en|volume=159|issue=7|pages=1413–1425|doi=10.1097/j.pain.0000000000001145|issn=0304-3959|via=}}
24. ^1 2 3 4 5 6 7 {{cite journal | vauthors = Bhatia SN, Ingber DE | title = Microfluidic organs-on-chips | journal = Nature Biotechnology | volume = 32 | issue = 8 | pages = 760–72 | date = August 2014 | pmid = 25093883 | doi = 10.1038/nbt.2989 }}
25. ^{{cite journal | vauthors = Pampaloni F, Reynaud EG, Stelzer EH | title = The third dimension bridges the gap between cell culture and live tissue | journal = Nature Reviews. Molecular Cell Biology | volume = 8 | issue = 10 | pages = 839–45 | date = October 2007 | pmid = 17684528 | doi = 10.1038/nrm2236 }}
26. ^{{cite journal | vauthors = Huh D, Hamilton GA, Ingber DE | title = From 3D cell culture to organs-on-chips | journal = Trends in Cell Biology | volume = 21 | issue = 12 | pages = 745–54 | date = December 2011 | pmid = 22033488 | pmc = 4386065 | doi = 10.1016/j.tcb.2011.09.005 }}
27. ^1 {{cite journal | vauthors = van Duinen V, Trietsch SJ, Joore J, Vulto P, Hankemeier T | title = Microfluidic 3D cell culture: from tools to tissue models | journal = Current Opinion in Biotechnology | volume = 35 | pages = 118–26 | date = December 2015 | pmid = 26094109 | doi = 10.1016/j.copbio.2015.05.002 }}
28. ^{{cite journal | vauthors = Soroush F, Zhang T, King DJ, Tang Y, Deosarkar S, Prabhakarpandian B, Kilpatrick LE, Kiani MF | title = A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction | journal = Journal of Leukocyte Biology | volume = 100 | issue = 5 | pages = 1027–1035 | date = November 2016 | pmid = 27190303 | pmc = 5069089 | doi = 10.1189/jlb.3ma0216-087r }}
29. ^{{Cite journal | vauthors = Tang Y, Soroush F, Deosarkar S, Wang B, Pandian P, Kiani MF |date= April 2016 |title=A Novel Synthetic Tumor Platform for Screening Drug Delivery systems |journal=The FASEB Journal |doi=10.1096/fasebj.30.1_supplement.698.7 |doi-broken-date= 2019-03-03 }}

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