Application Note
The PromoCell Cancer Stem Cell Medium
The PromoCell Cancer Stem Cell Medium has been designed to meet your requirements for the extended rial 3D tumorsphere culture.
This Cancer Stem Cell Medium supports the most commonly ud cancer stem cell lines in tumorsphere/mammosphere culture.
In contrast to the current adherent 2D culture of cancer cells, this type of 3D culture lectively exploits inherent
biologic features of Cancer Stem
Cells (CSC), such as anoikis resistance
and lf-renewal (Fig. 3). Continuous
proliferation is also supported during
rial passage of 3D tumorsphere cul-
tures (Fig. 2). Thus, this culture system
is also applicable for in vitro models of
metastasis.
PromoCell’s Cancer Stem Cell Medium
is ready-to-u and chemically defined,
providing a standardized culture devoid
of stimuli of uncharacterized origin.
This is a significant benefit in terms of
CSC which are a population of highly
responsive stem cells requiring reliable
and reproducible control of the lf-
renewal/differentiation axis.
The chemically defined PromoCell
Cancer Stem Cell Medium is suitable
for the cost-efficient and standardized
routine culture of tumorspheres/mam-
mospheres for a wide range of cell lines
(Tab. 1).
Fig. 1: Schematic flowchart of the TFE assay. Single cancer cells obtained by enzymatic dissociation of 3D tumorsphere cultures are plated by limiting dilution to one cell per well on 96-well plates. After an adequate incubation period, the wells originally containing the cells are analyzed for tumorsphere formation. Cells with stemlike properties are capable of forming a new tumorsphere derived from a single cell, while more restricted or differentiated tumor cells undergo anoikis. See the protocol on page 5 for details.
The TFE Assay
CSCs lack specific or universally applicable markers, so analysis and cha-racterization of the unique cancer cell subpopulations remain challenging. The most widely accepted methods there-fore rely on detection of specific func-tional cellular features. The tumorsphere formation efficiency (TFE) indicates the percentage of cells within a culture that are capable of forming a sphere from a
single cell (Fig. 1). Since this property is
only attributed to stemlike cells, the TFE
assay remains a valuable qualitative and
quantitative tool bad on exclusively
functional features of cells under lec-
tive culture conditions.
Originally derived by limiting dilution
neurosphere assays for detecting neu-
ral (cancer) stem cells, the TFE assay in
nonneuronal cancer cell cultures was
adapted as an universally applicable
technique. The neurosphere assay pro-
vides unique functional lectivity for
cells with stemlike properties and is a
uful tool for cell cultures of various
胃乃安胶囊other types of cancer. This TFE assay is
therefore particularly uful for explo-
ring and characterizing unidentified CSC
subpopulations without the need to rely
on unassured markers.
Tab. 1: List of cell types tested for rial passage with the PromoCell Cancer Stem Cell Medium.
Where rial passage is concerned, the culture media that had been established by then did not robustly allow for 3D sphere formation from cancer cells from other types of tumors. Indeed, this obstacle was due to the great he-terogeneity and variety of metabolic requirements of different types of cancer cells. The PromoCell Can-cer Stem Cell Medium, which sup-ports established cell lines of a wide variety of cancers, fills this remaining
gap. Enabling both rial passage and single cell-derived sphere formation, now all stem cell-related advantages of the 3D sphere culture technique are available for a broad range of cancers. Dependin
g on the culture conditions, the TFE can either remain fairly static or be distinctly dynamic. For example, in adherent rum-containing cultures of established cancer cell lines–standard culture conditions favoring differentia-tion–the TFE remains relatively stable at a low level. By contrast, the TFE may behave dynamically, especially after being switched from a standard culture to an environment that is more lective for stem cells. Under the conditions, sphere-forming cells may be gradually enriched (Fig. 2b).
Conquently, the TFE assay is a signifi-cant, cost-effective, and reliable in vitro
Differentiation potential Self-renewal Telomera activity Infinite lifespan
Stem cell
Mature cells with limited proliferative potential
Cancer stem cell (CSC)
Premalignant evolution
Progression
Tumor Secondary tumor
CSC - targeted therapeutic Metastasis
Benign cancer cells with limited proliferative potential (no tumor formation)
Application Note - Determination of the Tumorsphere Formation Efficiency (TFE)Fig. 3: Schematic overview on the origin, evolution and fate of cancer stem cells (CSC).
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女性性欲低Application Note - Determination of the Tumorsphere Formation Efficiency (TFE) 4
Background
“The term ‘malignancy’ refers to can-cerous cells that have the ability to spread to other sites in the body (me-tastasize) or to invade nearby (locally) and destroy tissues. Malignant cells tend to have fast, uncontrolled growth and do not die normally due to chang-es in their genetic makeup.
Malignant cells that are resistant to treatment may return after all detect-able traces of them have been removed or destroyed.” (Medline Encyclopedia)
History of cancer
Paleopathologic findings indicate that cancer has been recognized for millen-nia. The first written documentation of human cancer dates from 3000 BC, with the first description of metastasis going back to the year 50 AD [1]. More than 5000 years later, many types of cancer still remain incurable and prove fatal, particularly in cas of recurrence after emingly successful primary treatment. In 1863, almost 160 years ago, the pa-thologist Rudolf Virchow was the first to propo the model of cancer stem cells stating that ‘immature cells’ reprent the origin of cancer [2]. In 1959 Pierce introd
uced the term ‘cancer stem cell’ [3], while in 1997 Bonnet described a specific subpopulation of CSC as ‘can-cer driver cells’ in myeloid leukemia [4]. Recently, CSC have been identified in
various cancers including hematopoietic
malignancies and a range of solid tumors
[5].
Rearch and therapy for
malignancy
Cancer rearch is one of today´s ma-
jor rearch fields producing thousands
of publications every year. The most
frequently studied malignancies are
cancers of the breast, lung, liver, colon,
skin, pancreas and nervous system. Tra-
ditional therapeutic approaches aim to
eliminate as much of the tumor mass含糊的反义词
as possible by means of surgery, irra-
diation, chemotherapy and biologics.
H owever, accumulating evidence sug-
女孩写真gests the measures target the more
harmless, rapidly dividing cell mass of
the tumor and do not eradicate the pu-
tative root of the dia - CSC.
In the cancer stem cell model of tumors,
CSC are defined as a small subt of
malignant cells with the exclusive ability
to lf-renew and maintain the tumor.
They can differentiate into a heteroge-
neous mass of non-tumorigenic cancer
cell types, which usually constitute the
majority of the tumor [6]. It is clear in
this context that CSC, despite their ma-
lignant phenotype, share common hall-
marks of normal stem cells (Fig. 3), as-
signing extraordinary biologic potential
to the cells. The combined capa-
bilities account for one of the greatest
risks in the treatment of malignancies:
metastasis.
CSC are lf-sustaining and largely re-
营养不良吃什么sistant to anoikis, the term for apoptosis
induced by the detachment of adher-
ently growing cells from the extracel-
lular matrix. Therefore, CSC can pa-
小麦种植时间rate from the primary tumor, travel and
spread through the body where they
may form condary tumors (metasta-
s) in distant organs. Metastas may
develop quickly or over veral years
after emingly successful treatment
of the primary tumor. It is believed that
relap is caud by quiescent CSC that
are able to evade current therapeutic
regimens by using protective mecha-
nisms mediated by their stem cell prop-
erties.
It is clear that cancer rearch needs to
be realigned, especially when exploring
new clinical strategies to treat malig-
nancies. CSC are now considered to be
a new therapeutic target and it is be-
立春的含义是什么意思lieved that their elimination could lead
to permanent remission or even cure.
This might be achieved by direct eradi-
cation of the CSC or by specific adapta-
tion of CSC cell division from asymmet-
ric to symmetric leading to elimination
of the CSC population by blocking their
lf-renewal capabilities [6, 7]. For this
to be achieved, detailed characteriza-
tion is required.
method for indirectly measuring the CSC content of cultured cancer cells at a specific point of time and/or in a certain culture state. By way of example, the repeated concomitant determination of
TFE at different passages during rial
culturing of cancer cells in the PromoCell
Cancer Stem Cell Medium can indirectly
quantify the effects of this medium with
regard to the culture’s functional CSC
content.
Fig. 4: Tumorsphere culture of MCF-7 mammary carcinoma cells in the PromoCell Cancer Stem Cell Medium (C-28070) after 10 rial passages. The tumorsphere culture was subjected to rial passage every 9 days by enzymatic dissociation according to the
protocol. Robust tumorsphere formation was maintained during rial culture. See Fig. 1 for proliferation data.
5
Application Note - Determination of the Tumorsphere Formation Efficiency (TFE)Determination of the Tumorsphere Formation Effi-ciency (TFE)
TFE assay protocol
走亲戚的作文I. Materials
Tumorspheres (e application note “Tumorsphere Culture of Cancer
Stem Cells (CSC) with the PromoCell Cancer Stem Cell Medium” at /app-notes) Cancer Stem Cell Medium (C-28070) Phosphate buffered saline w/o Ca ++/Mg ++ (PBS, C-40232) DetachKit (C-41210) 96-well u-bottom suspension plates (e.g. Greiner Bio One, No. 650 185) 40 µm cell strainer Optional: multichannel pipet (100 µl)A) Generation of a single-cell suspension
This procedure largely corresponds (steps 1–5) to the subculture protocol of tumorsphere cultures, which routinely requires generation of a single-cell sus-pension.1. Collect the tumorspheres
Transfer the PromoCell Cancer Stem Cell Medium containing the tumorspheres into 15 ml conical tubes using a rological pipet.2. Gravity dimentation of the tumorspheres
Allow the spheres to ttle by gravity dimentation for 10 minutes at room temperature. Aspirate the supernatant, but leave approximately 200 μl in the conical tube. Do not aspirate the tumorspheres.3. Wash the tumorspheres
Repeat the dimentation (step 2 on the previous page) with an equal volume of PBS. Gently aspirate the PBS leaving approximately 200 μl in the conical tube. 4. Enzymatic digestion of the tumorspheres
Add 1 ml of trypsin-EDTA to the tumorspheres and incubate for 2–4 minutes at room temperature. Keep the spheres resuspended in the trypsin solution by pipetting up and down once every 30 conds. Avoid dimentation of the spheres.
Note: The optimal incubation time required to achieve complete dissociation in step A5 (below) must be empirically determined by the ur for each cell type. While 2–3 minutes will be optimal in most cas, tumorspheres of some cell types, e.g. MCF-7, may need longer incubation, especially in higher passages. If a completely defined dissociation process is preferred, a recombinant trypsin sol
ution may be ud as an alternative dissociation reagent while following the supplier’s instructions.5. Break down remaining cell aggregates
Pipet the spheres up and down 10–20 times using a 1000 μl pipet tip to gene-rate a single-cell suspension. Aspirate the cell suspension in the normal way but slightly tilt the pipet tip at the bottom of the tube when expelling the cells. The generated shear forces facilitate the breakup of any residual cell aggre-gates. Perform a visual check to confirm that no large cell aggregates remain. Immediately after trituration, add twice the volume of Trypsin Neutralization Solution (TNS).
