Improving stem cell purity using microfluidics

Transcription

1 Improving stem cell purity using microfluidics Lisa A. Flanagan, Ph.D. Cal-IT2 Igniting Technologies University of California, Irvine November 10, 2011

2 Stem cell purity for regenerative medicine cell differentiation is necessary but generates heterogeneity Hematopoietic Stem Cells NIH molmed.lu.se

3 Neural stem cells: restricted progenitors glia:

4 Cell sorting by dielectrophoresis Dielectrophoresis (DEP) DEP force is due to the interaction of a particle s dipole and the spatial gradient of a nonuniform AC electric field F dep = p E p: the particle s dipole moment : the gradient of the electric field Advantages: label-free unbiased Voldman, 2006

5 Dielectrophoresis - how does it separate cells? cell velocity (microns/sec) frequency (MHz) Positive DEP Negative DEP Pethig et al., 2002 electrode Previous separations using DEP: breast cancer cells (MCF) +/- neu oncogene human leukemia cells out of blood human breast cancer cells from blood normal from malaria-infected blood cells

6 Microfluidics and DEP force (MF3 Center) Collaboration with Dr. Abe Lee s lab Lu et al., submitted

7 Microfluidic DEP device: mouse neural stem cells Flanagan et al., Stem Cells 2008

8 DEP distinguishes stem cell differentiation and fate potential DEP trapping curves differentiated cells and NSPCs

9 Cell characteristics detected by DEP dielectric properties at lower frequencies are dominated by membrane characteristics neural stem cell neuronal progenitor immature neuron

10 Label-free detection of stem cell differentiation Lu et al., submitted

11 DEP isolates progenitors with less toxicity than FACS Amanda Dickson, Jami Nourse, Javier L. Prieto

12 Current cell separation technology (FACS) vs. DEP Fluorescence-activated cell sorting (FACS) DEP-based cell sorting - size: ~4-6 feet space needed - cost: ~$500,000 or more - expensive, complicated lasers - requires labels/antibodies - one per institution - size: fits on ~2 feet bench space - cost: ~$500 or less - inexpensive function generator - label-free, no need for antibodies - one per lab

13 Neural stem cells: DEP and cell purity DEP in microfluidic devices provides a label-free and non-toxic way to distinguish and isolate restricted progenitors for study or transplantation

14 Acknowledgements and Collaborators Flanagan Lab (Neurology Dept.) Jente Lu Jami Nourse Amanda Dickson Steve Marchenko Minjee Do Abe Lee (BME Dept. and MF3 Center) Javier Lopez-Prieto Lisen Wang John Collins UCI Sue and Bill Gross Stem Cell Research Center INRF Support NIH CIRM SET-Squared CDA Roman Reed Research Award

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16 Incorporation of DEP into analysis of stem cells DEP-based isolation of progenitor cells DEP and FACS isolation of progenitor cells analysis of cells characterization of protein expression FACS-based isolation of progenitor cells cell identification in vitro and in vivo identification of unique markers

17 Central nervous system neural stem cells

18 Neural stem cells: restricted progenitors transplant

19 Unique cell-surface epitopes for FACS? neuron PSA-NCAM CD133 or LeX + CD24 some CD133-negative some A2B5 glioblastoma CD133, CD24 PSA-NCAM CD24 A2B5? CD133 LeX (CD15, SSEA1) SSEA4 astrocyte A2B5 Catch-22: no markers to isolate cells, so how isolate cells to get markers? could there be an alternative and complementary approach?

20 DEP based cell trapping DEP forces can attract cell subpopulations to electrode arrays Das et al., 2005

21 E12 mouse neural stem cells are more neurogenic differentiated neurons derived from neural stem cells isolated from the developing cortex

22 Human neural stem cells differ in fate potential SC27 SC23 Labeed et al., submitted

23 DEP is not toxic for neural stem cells cell viability cell proliferation trypan blue MTT LDH EdU incorporation cell cycle analysis mouse and human NSPCs caveat - long term exposure to negative DEP can be toxic

24 Membrane capacitance (Cspec) correlates with neurogenic potential of mouse and human NSPCs mouse NSPCs human NSPCs E12 more neurogenic E16 less neurogenic SC27 more neurogenic SC23 less neurogenic ** p<0.01 Labeed et al., submitted

25 Membrane capacitance (Cspec) reflects neurogenic potential of human NSCs changing neurogenic potential over time neurogenic potential of different human NSCs Cspec neuron generation Labeed et al., submitted

26 In contrast, membrane conductance does not correlate with neurogenic potential Labeed et al., submitted

27 Sorting strategies using positive DEP electrodes channel outlets cells well controlled ~10,000 cells/trap high throughput ~10,000 cells/sec Abe Lee, Javier L. Prieto, Jente Lu

28 DEP-based sorting enriches neurogenic progenitors undifferentiated NSPCs DEP sorting differentiate to determine fate Jami Nourse, Javier L. Prieto, Amanda Dickson

29 DEP band sorting of neuronal progenitors Jami Nourse, Javier L. Prieto

30 NSPC membrane glycosylation contributes to dielectric properties Jami Nourse, Mike Demetriou

31 Dielectrophoresis to separate cells 100 crossover frequency cell velocity (microns/sec) 50 0 frequency (MHz) Positive DEP Negative DEP Pethig et al., 2002 electrode

32 Crossover frequency and neurogenic potential of human NSCs changing neurogenic potential over time neurogenic potential of different human NSCs crossover frequency neuron generation can we use DEP to separate NSCs by fate potential? Labeed et al., submitted

33 transplanted NSCs: enhance survival specify differentiated cell fate combination with other matrices isolated NSCs: transplantation analysis of membrane differences NSC lineage tree Summary and Future Directions microenvironment 3D substrates for cells purity distinguish cell subpopulations salmon fibrin: neurite outgrowth improve functional recovery enhance caudal fibers DEP of NSCs: label free identify progenitors by fate potential isolate biased progenitors