Life-like preservation and TEM visualization of the glycocalyx reveals that it is substantial in vitro

E. E. Ebong, Frank P. Macaluso, David C. Spray, J. M. Tarbell

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function, due to findings that GCX in vitro may be substantially thinner than in vivo. The assessment of thickness differences is complicated by GCX collapse during dehydration for traditional electron microscopy. We measured in vitro GCX thickness using rapid freezing/freeze substitution transmission electron microscopy (RF/FS-TEM), taking advantage of high spatial resolution provided by TEM and the capability to stably preserve the GCX in its hydrated configuration by RF/FS. Bovine aortic and rat fat pad endothelial cells (BAEC and RFPEC) were subjected to conventional- or RF/FS-TEM. Conventionally preserved BAEC GCX was ∼0.040 μm in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ∼11 μm and RFPEC GCX of ∼5 μm. RF/FS-TEM also discerned GCX structure and thickness variations due to heparinase III enzyme treatment and extracellular protein removal, respectively. Immunoconfocal studies confirmed that the in vitro GCX is several microns thick and is comprised of extensive and well integrated heparan sulfate, hyaluronic acid, and protein layers. New observations by RF/FS-TEM reveal substantial GCX layers on cultured EC, supporting their continued use for fundamental studies of GCX and its function in the vasculature.

Original languageEnglish (US)
Title of host publication2011 IEEE 37th Annual Northeast Bioengineering Conference, NEBEC 2011
DOIs
StatePublished - 2011
Event37th Annual Northeast Bioengineering Conference, NEBEC 2011 - Troy, NY, United States
Duration: Apr 1 2011Apr 3 2011

Other

Other37th Annual Northeast Bioengineering Conference, NEBEC 2011
CountryUnited States
CityTroy, NY
Period4/1/114/3/11

Fingerprint

Freezing
Visualization
Transmission electron microscopy
Substitution reactions
Endothelial cells
heparitinsulfate lyase
Proteins
Hyaluronic acid
Heparitin Sulfate
Hyaluronic Acid
Oils and fats
Dehydration
Cell culture
Electron microscopy
Rats
Enzymes
Fats

ASJC Scopus subject areas

  • Bioengineering

Cite this

Ebong, E. E., Macaluso, F. P., Spray, D. C., & Tarbell, J. M. (2011). Life-like preservation and TEM visualization of the glycocalyx reveals that it is substantial in vitro. In 2011 IEEE 37th Annual Northeast Bioengineering Conference, NEBEC 2011 [5778556] https://doi.org/10.1109/NEBC.2011.5778556

Life-like preservation and TEM visualization of the glycocalyx reveals that it is substantial in vitro. / Ebong, E. E.; Macaluso, Frank P.; Spray, David C.; Tarbell, J. M.

2011 IEEE 37th Annual Northeast Bioengineering Conference, NEBEC 2011. 2011. 5778556.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Ebong, EE, Macaluso, FP, Spray, DC & Tarbell, JM 2011, Life-like preservation and TEM visualization of the glycocalyx reveals that it is substantial in vitro. in 2011 IEEE 37th Annual Northeast Bioengineering Conference, NEBEC 2011., 5778556, 37th Annual Northeast Bioengineering Conference, NEBEC 2011, Troy, NY, United States, 4/1/11. https://doi.org/10.1109/NEBC.2011.5778556
Ebong EE, Macaluso FP, Spray DC, Tarbell JM. Life-like preservation and TEM visualization of the glycocalyx reveals that it is substantial in vitro. In 2011 IEEE 37th Annual Northeast Bioengineering Conference, NEBEC 2011. 2011. 5778556 https://doi.org/10.1109/NEBC.2011.5778556
Ebong, E. E. ; Macaluso, Frank P. ; Spray, David C. ; Tarbell, J. M. / Life-like preservation and TEM visualization of the glycocalyx reveals that it is substantial in vitro. 2011 IEEE 37th Annual Northeast Bioengineering Conference, NEBEC 2011. 2011.
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