DiscoveryBioMed, Inc. Human ADPKD Primary Cell Cultures
Spring 2016
Updated 3.9.16
DBM, Inc. acted as a Cell Culture and Engineering Core D for the Baltimore
PKD Center in the first 5 years of the NIH NIDDK P30 PKD Research and Translational Core Centers
program. These efforts have focused on the generation of mouse PKD vs WT renal epithelial cell lines
(see a separate document on what cultures and cell lines are available).
There were as many requests from Research Base investigators of our P30 and beyond about the
availability of human cell cultures and cell lines with relevance to PKD. For our own interests and to
complement our efforts for the Baltimore PKD Center, we ramped up this parallel effort. We collaborate
with this Center and now the Mayo PKD Center and the Kansas PKD Center on the further development
of these primary cultures from both human ADPKD donor kidneys and from normal kidney cells and
tissues. Genotyping information on the huADPKD cells is becoming available with our primary culture
offerings.
Source Material: DBM, Inc. has multiple commercial vendors/exchanges where it has protocols to obtain
fresh human normal or ADPKD kidney tissue. DBM also has multiple commercial and academic sources
for primary human normal and ADPKD kidney cell cultures. The Baltimore P30 PKD Center and the Mayo
P30 PKD Center will also be sources of this tissue material going forward and will collaborate with DBM
on the generation of these human cells and on their genotyping for PKD1 and PKD2 gene mutations,
respectively. All collaborators are well respected PKD academic investigators.
Cell Culture and Immortalization Critical Path: Immortalized normal and diseased human kidney cells are
becoming available going forward. Every immortal clonal cell line has its origins in primary cell cultures
established from fresh cellular or tissue material. One then exposes the best primary cell cultures to
immortalization constructs (genetic material designed to render primary cells to be more “longer-lived”
or immortal). What emerges next is mixed immortalized cell cultures. These cultures can be useful as
well. One cryopreserves the primary and mixed immortalized cultures as a ‘fall back’ in case cloning
presents difficulties. Finally, cloning of mixed immortalized cultures ultimately yields immortal clonal cell
lines – normal kidney or ADPKD kidney. DBM utilizes multiple and different methods of immortalization
to ensure results.
This Critical Path is presented in simplest terms below:
Primary Human Cell Cultures > Mixed Immortalized Cell Cultures > Immortal Clonal Cell Lines
(Mixed Population of Cells) >
(Mixed Population of Cells)
>
(Clonal Cell Line)
Huge Unmet Need: There remains a huge unmet resource need (if not a complete lack of resources) of
any novel, normal human kidney cell lines. Based upon our early efforts, DBM believes it knows why so
few (if any) have emerged that are non-carcinoma cell lines. There also remains a complete lack of
normal human kidney cell lines as well as human ADPKD kidney cell lines that maintain disease
characteristics, in vivo-like phenotype, and robust propagation. Again, we believe that we know why and
our lessons learned to date seek to solve this problem and to provide these biologically-relevant and
disease-relevant human cell platforms to the renal biology and physiology research community and with
basic knowledge of characteristics and best growth conditions.
This effort remains a work in progress and we are still accumulating best practices – users can help us
with this cause in relaying back what primary cultures and cell lines work for them and why.
Lessons Learned to Date: Permeable filter support-driven culture (for both epithelial and non-epithelial
cells) has been the most useful method to date for our human and mouse WT/normal versus
PKD/ADPKD cell platform creation efforts. Tissue culture plastic-driven culture has not been “value add.”
With respect to normal human kidney epithelial cells, DBM has been able to procure or create primary
human cell cultures that maintain for multiple passages before slowing growth and dying. DBM has also
established successfully primary human normal kidney epithelial mixed immortalized cell cultures.
Despite these efforts, their growth begins to slow. DBM can clone individual islands of clonal immortal
normal human kidney epithelial cells; however, they sit on the permeable filter support and growth
arrest. They do not die – they just do not grow.
Permeable filter support-driven culture has helped mitigate these issues but the cells still slow and
eventually stop growth. So, we have applied a critical lesson learned from the recent processing of a
human ADPKD donor kidney. The amount of fibrosis within this end-stage kidney (removed ahead of a
renal transplant) was dramatic. In hindsight and for new tissue to come, the tough and extensive
connective tissue within these diseased kidneys will also be processed to yield precious human ADPKD
fibroblast cell cultures. We will also grow normal human kidney primary cultures from fresh kidneys or
fresh primary cultures of low passage to yield primary cultures of normal human kidney fibroblasts.
Existing routine methods (used by most) are usually designed to remove pesky fibroblasts that overgrow
epithelial cell cultures – DBM has learned that it is essential to also cultivate and cryopreserve normal
and ADPKD fibroblast cell cultures. Why? When fibroblasts are present in the mixed populations of
primary cultures and mixed immortalized cell cultures, the epithelial cells continue to proliferate. When
you clone out the epithelial cells and eliminate the presence of fibroblasts, they slow and stop. This is
also true of keratinocytes without or with dermal fibroblasts; the same is true for GI epithelial cells
(enterocytes, gall bladder, bile duct) without or with supportive cells like fibroblasts. Along these lines,
DBM is experimenting currently with fibroblast co-culture support and fibroblast-conditioned medium
support (fresh vs. refrigerated vs. frozen). When fibroblast-conditioned medium is fed to epithelial cellenriched cultures, proliferation renews or accelerates. This remains a study in progress in both 2D and
3D culture.
Current Hypothesis: We hypothesize that active co-culture with fibroblasts will be the most effective in
bringing out the best in WT/normal and PKD/ADPKD epithelial cell lines, followed by freshly collected
fibroblast-conditioned medium. We are currently testing fresh conditioned medium versus refrigerated
fibroblast-conditioned medium for differences. We do not yet know if frozen fibroblast-conditioned
medium will be effective or not but it will be tested.
Examples of What DBM Has to Date: Based upon consultation with multiple PKD researchers and based
upon prior experience of Dr. Schwiebert of DBM, DBM has processed recently its first 4 human ADPKD
donor kidneys into primary cultures. Individual cyst-derived primary cultures and multicystic tissue
cluster-derived primary cultures are available from each kidney and have been characterized for growth
in both 2D and 3D Biogel based cultures.
Virtually pure cystic epithelial cell cultures derived from a single cyst – DBM seeks
to dissect away the surrounding tissue from the fluid-filled cyst before removal
of cyst fluid, mincing of cyst walls and digestion of cyst wall tissue material
Segment of a Human
ADPKD Donor Kidney
“Cyst 3” huADPKD cystic epithelial cells growing
out of a cyst wall fragment
“Cyst 3” huADPKD cysts formed within 3D Matrigel
culture
Cyst 1
Cyst 2
Cyst 3
huADPKD fibroblasts
Cyst 4
Cyst 5
Cyst Wall 6
Cyst Wall 7
Figure 1: Example of Single Cyst-derived
Primary Culture and 2D and 3D Growth
Formats in DBM RenaLife Hybrid Media.
See text for details and methods.
Arrows mark huADPKD cysts
(out of focus)
Single Cyst-derived Primary Culture
Single
mature
huADPKD
cyst in
Matrigel
huADPKD fibroblast latticework
between huADPKD cysts in Matrigel
Arrows mark huADPKD cysts
(out of focus)
Deliverables: DBM has single cyst-derived ADPKD epithelial cell primary cultures and multi-cystic tissuederived epithelial cell primary cultures to provide to investigators. DBM can also provide primary ADPKD
fibroblasts for co-culture and/or fibroblast-conditioned medium for cultivation of fibroblast-conditioned
medium in your laboratory.
Normal Mixed Cortical (Lifeline Cell Tech)
Normal Mixed Medullary (Lifeline Cell Tech)
Figure 2: Examples of Cystogenesis in 3D
Matrigel Cell Culture Format with DBM
RenaCyte Medium for DBM Primary
huADPKD Cystic Cell Cultures (see above)
and Primary Normal Human Renal
Epithelial Cells (at left). Primary normal
human kidney cells form cysts in 3D, more
so with mixed cortical cell cultures and
rarely with mixed medullary cell cultures.
Tubule or duct-like structures are observed
in normal cultures.
DBM has a specialty medium, DBM RenaCyte medium, that it recommends and will sell through our
Online Store.
Both the cells and the media are tested and free of pathogens.
Please contact DBM’s Dr. Erik Schwiebert to negotiate a bundled shipment of cells, medium and ‘know
how’ to get you started. [email protected]