CORE C: INTEGRATED PHYSIOLOGY


Director, Nicholas C. Zachos, Ph.D.
nzachos1@jhmi.edu
410-614-0128

 

 


Associate Director, Ming Tse, Ph.D.
mtse@jhmi.edu
410-955-9681

 

 

GOALS

The Integrated Physiology Core provides multiple services and serves as a centralized source for equipment and reagents to enhance studies of epithelial biology in the gastrointestinal tract.

SERVICES

  • Murine and human gastrointestinal organoids
  • The Integrated Physiology Core provides training for establishing, expanding and maintaining mouse and human enteroid cultures. Core C instructs Center investigators how to isolate intestinal crypts from mouse and human tissues (i.e. biopsies or resections), seed into the 3D matrix (i.e. Matrigel), propagate and split cultures, cryopreserve cultures (to establish their own biobanks), and recover cultures from 3D for biochemical studies. The service allows Center investigators to develop this model within their own laboratories. The Core also prepares conditioned media containing the critical growth factors for investigators who study these organoid cultures. Fresh conditioned media is prepared weekly and provided to the Research Base on a fee-for-service basis at >90% cost savings.
  • Histology
  • The Core can process (fresh or frozen), embed (paraffin or OCT), section and stain tissues samples, including animal models and humans. The Core offers a variety of stains including hematoxylin and eosin, Masson’s Tri-Chrome Blue, Periodic Acid Schiff, Von Kossa, and Sirius Red. Paraffin and frozen sections may also be archived by the Core for future studies or collaborations.
  • Ussing chamber/Voltage clamp
  • The Core provides equipment and training for the measurement of active ion transport in cultured polarized epithelial cell models, ex vivo mouse and human intestine and human enteroid monolayers. Dr. Tse provides training for the equipment and advice for experimental design, including methods of calculation, quality control, and data interpretation. Applications include measurement of short circuit current, trans-epithelial resistance (TER), dilution potential, and paracellular dextran flux.

EQUIPMENT

• Physiological Instruments 6-Channel Voltage Clamp Connected to Easy Mount 6-Channel Ussing Chamber System
• World Precision Instruments EVOM2, Epithelial Voltohmmeter for TER in 24-well and 96-well formats

Multiplex Cytokine ELISA
The Core has established a multiplex ELISA system capable of analyzing various cytokines and/or chemokines simultaneously from the same serum or tissue samples obtained from mice and/or humans.

Bio-Rad 200 Plex
• Luminex-based multiplex ELISA systems

TRAINING

The Core provides extensive training for establishing and maintaining murine and human gastrointestinal stem cell-derived organoids.

FEES

ServiceAssociate MembersFull MembersNon-MembersIHC World
Tissue processing and embedding$4.00/block$4.50/block$5.00/block$6.00/block
FFPE Sections
Slide box (100 slides)
$1.50/slide
100% cost-pay
$2.00/slide
100% co-pay
$2.50/slide
$10/box
$4.00/slide
$12/box
Cryosections$1.50/slide
$2.00/slide
$5.00/slide
$10/slide
H&E Staining
Other stains
$2.00/slide
$2.00/slide
$2.50/slide
$2.50/slide
$3.00/slide
$5.00/slide
$5.00/slide
$12.00/slide
Enteroid training
Enteroid media
100% cost offset
$2.00/mL
$250 ($250 cost offset)
$3.00/mL
$500
$3.50/mL

NEW SERVICES IN DEVELOPMENT

The Core will continue to develop services that fill gaps in the research of our Research Base. The greatest perceived needs relate to human enteroids, which are being used more and more by our Investigators to address questions of normal and abnormal intestinal function and disease. The following are the areas that are being developed by Core C:

(1) Development of Enteroid Co-Culture

Enteroids derived from isolated crypt stem cells are comprised of all epithelial cell types (Naabsorptive and Cl– secretory enterocytes, Paneth, Goblet, and enteroendocrine cells) but lack many other cells types that are involved in normal gut function and disease. These include myofibroblasts, vascular cells, enteric nerves, immunologic and inflammatory cells. The laboratory of Dr. P. Pasricha is developing the methodology to isolate and culture mouse and human sub-mucosal neural stem cells that can be grown in a way that they interact with enteroids. Currently, the Pasricha lab in collaboration with the Donowitz laboratory has observed that partially and fully differentiated mouse enteroid monolayers can be grown to interact with murine submucosal nerves in a way that alters electrogenic anion secretion and appears to duplicate findings of intact intestine in which ~50% of cholera toxin and rotaviral diarrhea is neutrally mediated. The laboratory of Dr. Zachos has established the methodology for co-culturing human monocyte-derived macrophages, neutrophils, dendritic cells, and differentiating ileal enteroids to be enriched with Microfold (M) cells (See Fig. 1).

Core C Figure 1 Part A
Figure 1: Human enteroid-immune cell co-cultures and human ileal enteroids differentiated to express Microfold (M) cells. A) Innate immune responses to Enterotoxigenic E. coli (ETEC) infection of human enteroid monolayers co-cultured with primary human macrophages (MΦ) 8.
Core C Figure 1 Part B
Figure 1: Human enteroid-immune cell co-cultures and human ileal enteroids differentiated to express Microfold (M) cells. B) Primary human neutrophils transmigrate across enteroid monolayers in response to luminal IL-8 exposure.
Core C Figure 1 Part C
Figure 1: Human enteroid-immune cell co-cultures and human ileal enteroids differentiated to express Microfold (M) cells. C) Human ileal enteroids can be differentiated to express Microfold (M) cells. 5 day differentiated (i.e. no Wnt3a, no R-spondin-1) human ileal monolayers do not express M cells (“Control”); however, differentiation with RANKL and TNF- will induce expression of M cells (indicated by glycoprotein-2 (GP-2; M-cell specific marker) staining in red). White = actin. Human ileal M cells exhibit classic apical surface effacement as demonstrated by scanning electron microscopy similar to that observed in the in vivo human Peyer’s patch.

He will build upon established methods to co-culture additional innate immune cells (e.g. intraepithelial lymphocytes) with human enteroids/colonoids. The methods for co-culturing human macrophages and neutrophils are published. Additional co-culture protocols are being developed with non-Core Center resources, but the methods will be shared and taught to Core Center Investigators starting in Core Center Year 11.

(2) Development of “Gut-on-Chip”

Enteroids as currently studied, even as monolayers on permeable supports, only partially mimic the normal state.  Addition of physical forces to which the intestine is normally exposed, including luminal flow (mimicking the large volume exposed to lumen of normal intestine), basolateral flow (mimicking blood flow), and wall stretch and contraction (mimicking peristalsis) alter gene expression, cell height and differentiation when studied in Caco-2 cells and human enteroids. Through a previously established collaboration with Emulate, Inc, Core C has obtained the equipment necessary to culture human enteroids in an automated “gut-on-chip” platform. The Donowitz laboratory successfully demonstrated that this system could be used by a laboratory separate from the designing laboratory. The lab’s results showed an increased rate of epithelial differentiation and maturation of human enteroids when compared to enteroids cultured on Transwells. The Emulate equipment will be offered as a new Core Service with uncertain usage due to the chip expense. Training will be provided by a post-doctoral fellow from the Donowitz lab who has used the Emulate system extensively. This service is being offered starting towards the end of Year 10 of Core Center funding (delayed due to COVID-19) and a two-year trial period has been designated to determine the demand for the service.

(3) Human Enteroid Models of Normal GI Physiology and GI Diseases

Recent studies have provided strong evidence that human intestinal stem cell-derived enteroids/colonoids can recapitulate various aspects of normal GI physiology as well as epithelial changes that are characteristic of multiple GI disorders. For example, goblet cell numbers are altered in IBD and this phenotype can be observed in enteroids obtained from IBD patients with active disease. In addition, human enteroids from IBD patients retained altered cytokine profiles when compared to enteroids derived from healthy subjects (See Fig. 2). In order to meet the growing needs of the Research Base for use of human enteroids, Core C is working together with Core D to store and make available several normal enteroid lines from each intestinal segment as well as enteroids from patients with GI disease, that now includes Crohn’s ileitis and colitis, active and inactive ulcerative colitis, IBS-D, primary bile acid diarrhea, amyloid of the intestine, along with several other disorders. Aliquots are provided for individual laboratories to expand and maintain the enteroids. Enteroids are made available only to Core Center Investigators. This Core Service has just begun being offered and will be expanded during Year 11 of funding. Currently, 640 aliquots of 60 established enteroid lines (from each segment of the small intestine and colon) from healthy donors and several IBD enteroids have been transferred to the Translational Research Enhancement Core.

Core C Figure 2
Figure 2. Human colonoids derived from Ulcerative colitis (UC) patients are phenotypically distinct from healthy controls. A) Human colonoids were established from healthy donors (n=3) and from inflamed (“INF”) and pathologically normal (“N-INF”) regions of the colon from the same UC patient (n=4 total UC patients). Basolateral media was collected from healthy and UC colonoid monolayers that were differentiated for 5 days. MCP-1 and IL-8 levels were quantitated by MSD multiplex ELISA and elevated from both active and inactive UC colonids. * p<0.01. Yu and Zachos, unpublished. B) Inflamed (“INF”) colonoids from UC patients had significantly impaired growth rates compared to non-inflamed (“N-INF”) or healthy controls. C) Barrier function, as determined by transepithelial electrical resistance (TER) measurements on colonoid monolayers differentiated (DF) for up to 3 days, was different among groups. In both non-differentiated (blue bars) and differentiated (yellow bars) states, inflamed (“INF”) colonoids had higher TER values compared to non-inflamed (“N-INF”) colonoids or normal, healthy controls. ns=not significant. Zachos, unpublished.