Mouse ES Cell Culture

Mouse Pluripotent Stem Cells

Millipore provides the largest and most comprehensive range of products for your mouse ES cell culture needs.

Highlights of this Product Range Include:

ESGRO® mLIF Medium Supplement for maintaining undifferentiated ES cells
ESGRO Complete™ cell culture system, a defined serum-free system for the maintenance and derivation of mouse ES cell lines in the absence of FBS and feeder cells
RESGRO™ Culture Medium for the rescue of partially differentiated ES cell lines and improved ES cell derivation
B6-White ES cell line, the first commercially available C57BL/6 tyrc-2J albino line that allows for rapid coat-color determination of successful chimerism in C57BL/6 mice
Range of ES cell lines derived from different mouse strains
Mitotically inactivated and antibiotic resistant EmbryoMax PMEF feeder cell lines
EmbryoMax ES cell qualified reagents including DMEM, FBS and media supplements
EmbryoMax media and reagents for mouse embryo culture

Millipore’s range of ES cell-qualified reagents provides researchers with convenient and cost effective solutions for the reliable culture of ES cells. These products negate the need for researchers to screen lots of media, reagents and serum, thus delivering significant cost and time savings. Researchers can use the EmbryoMax range of products with confidence, as the same extensive validation processes behind our class-leading ESGRO products are used to qualify these new products for ES cell culture.

Millipore cell culture protocols include the in vitro culture of murine ES cells using the EmbryoMax range of ES cell qualified products with ESGRO mLIF medium supplement, as well as feeder-free and serum-free ES cell culture using the ESGRO Complete line of products. Also published are methods for improving the efficiency of existing ES cell lines (ES cell rescue) and derivation of new ES cell lines using RESGRO Culture Medium and ESGRO Complete cell culture system.

Embryonic Stem Cell Culture

ESGRO mLIF Cell Culture Supplement

Consistent inhibition of ES cell differentiation
Savings in time and increased convenience
Minimization of mycoplasma contamination
No batch to batch variation

The rapid development of gene knockout technology has provided an effective tool for the analysis of gene function. Critical to this technology has been the ability to isolate and culture embryonic stem (ES) cells in vitro. Derived from the inner cell mass of early mouse embryos, ES cells contribute to all tissues including germline.

The development of efficient procedures for the in vitro maintenance of the pluripotent phenotype of ES cells has been crucial to the success of gene targeting experiments. The use of the novel ES cell culture reagent ESGRO mLIF Medium Supplement for the in vitro maintenance of undifferentiated Murine ES cells is well established.

Exclusive to Millipore, ESGRO mLIF Medium Supplement contains the recombinant cytokine mLIF at an optimized concentration and activity. When used for the in vitro culture of murine ES cells, ESGRO supplement can replace the need for fibroblast feeder layers or medium conditioned by various other cell types.

ES (R1) cell colonies (10x) cultured using ESGRO mLIF Medium Supplement (1000 units/mL), in the absence of a Primary Mouse Embryonic Fibroblast (PMEF) feeder layer (left) and with PMEF feeders (right). In either the absence or presence of a PMEF feeder layer, a well-rounded ES cell colony morphology is seen in both examples due to ESGRO supplement acting to inhibit cell differentiation.

ESGRO Complete™ Serum-Free Cell Culture System

Feeder- and serum-free culture system for the successful propagation of mouse embryonic stem cells at clonal density while maintaining pluripotency
Enables mouse ES cell line derivation in a defined medium
Germline transmission is comparable to serum- supplemented medium

[insert hyperlink] ESGRO Complete Clonal Grade Medium is an innovative serum-free cell culture medium, which for the first time enables mouse ES cells to grow from clonal densities without the use of fetal bovine serum or feeders. This defined culture medium supports undifferentiated ES cell expansion and growth equal to that of serum-supplemented medium. It is suitable for mouse ES cell derivation, transfection and selection, in addition to general cell passaging and growth.

Traditionally, the culture of mouse ES cells has involved the use of serum supplemented basal medium and primary mouse embryo fibroblast (PMEF) feeder cell layers and/or LIF. This combination of factors was essential for maintaining mouse ES cell self- renewal and pluripotency. Recently, it has been shown that the use of BMP4 in conjunction with LIF replaces the need for serum and feeder cells for ES cell self-renewal and preserves multilineage differentiation, chimera colonization and germline transmission properties (Ying Q.L. et al., 2004, Cell).

ESGRO Complete Clonal Grade Medium is a complete medium containing BMP4 and LIF that supports mouse ES cell line derivation and maintenance. The resultant ES cell lines have normal karyotype, result in chimeric offspring and maintain germline competency at rates comparable to conventional methods. This novel medium allows the study of stem cell and developmental biology, performance analysis of gene targeting experiments, and the produc- tion of animal models for research without the uncer- tainty that comes from growing the cells in multiple, unknown and variable factors.

To confirm pluripotency of ES cells after many pas- sages in ESGRO Complete Clonal Grade Medium, cells were stained for alkaline phosphatase, Oct-4 and Nanog, and subjected to karyotypic analysis (not shown) and germ line competency. Cells have different growth characteristics than in serum being tighter and more closely packed together with less differentiation. Cells can be successfully passaged, frozen and thawed while maintaining their pluripotency with a normal karyotype and germ line competency.

A. E14Tg2a murine ES cells are plated at clonal density (1000 cells/10 cm dish in 10 mL of media). Media is changed at day 3. After 5 days, cells are fixed and stained for Alkaline Phosphatase. Colonies formed in 10% FCS without feeder layer tend to be more spread out and flatter in morphology, thus appearing larger in comparison. Some large colonies did not stain positive for Alkaline Phosphatase. B. Colonies formed in Clonal Grade Medium were more compact and uniform in shape. Colonies were generally smaller than in 10% FCS. All colonies stained strongly for Alkaline Phosphatase.

ESGRO Complete Clonal Grade Medium
Immunostaining of mouse ES cells grown in ESGRO Complete Clonal Grade Medium. From left to right for the top panel(A): phase contrast, Hoechst staining and Oct-4 staining. From left to right for the bottom panel (B): phase contrast, Hoechst staining and Nanog staining

RESGRO Culture Medium

RESGRO Culture Medium is a complete cell culture medium that can be utilized to complement traditional murine Embryonic Stem (ES) cell culture media containing ESGRO mLIF Medium Supplement. In contrast to traditional medium, RESGRO Culture Medium is recommended for a number of specialized applications including rescue, derivation and expansion of murine ES cells.

Rescue of Established ES Cell Lines

RESGRO Culture Medium has the capacity to rescue established ES cell lines that have started drifting and either generate low percentage chimeras or have lost germline transmission capability. ES cell differentiation, which is not visible with traditional medium, will become recognizable when using RESGRO Culture Medium. After two passages, a clear difference is seen between differentiated and undifferentiated ES cells, at which time undifferentiated cells can be removed by sub-cloning. ES cells can continue to be cultured in RESGRO medium or in medium containing ESGRO supplement.

The Derivation of New ES Cell Lines

RESGRO Culture Medium enables the efficient derivation and maintenance of ES cell lines from several inbred mouse strains, including certain strains that were previously considered to be non-permissive for ES cell derivation. A recent study demonstrated that RESGRO allowed the improved derivation of ES cell lines from 5 inbred strains other than 129, including FVB, a strain previously considered to be non-permissive for ES cell derivation, C57Bl/6N, BALB/c, 129/SvEv and DBA/2N mouse strains.

EmbryoMax® ES Cell Qualified Products

The EmbryoMax range of ES cell-qualified reagents provides researchers with a convenient and cost effective solution for the reliable culture of ES cells. These products remove the need for researchers to screen lots of media, reagents, and serum, thus delivering significant cost and time savings. Researchers can use this new range of products with confidence, as the same extensive validation processes behind our class-leading ESGRO products are used to qualify these new products for ES cell culture.

Improved Efficiency of Murine ES Cell Lines Using RESGRO Culture Medium

ES Cell LIne	Medium & Method Used	Number of Embryos Transferred	Number of Pups Born	Number of Chimeras Born	Percentage Chimerism
129SvEv Wild-type done	Traditional medium Diploid aggregation	40	28	4	1 X 2% 1 X 5% 1 X 10% 1 X 50%
129 SvEv Wild-type done	RESGRO medium Diploid aggregation	106	25	25 11 died	1 X 10% 1 X 90% 12 X 100%

Efficiency of ES Cell Derivation and Germline Competence with RESGRO Culture Medium

Mouse Strain	Blastocysts Cultured	Established ES Cell Lines		No. Germline Competent ES Cell LInes / No. ES Cell Lines Cultured
	(n)	(n)	(%)
C57Bl/6N	35	18	51	10/11
FVB/N	20	8	40	6/9
BALB/c	34	15	44	7/7
129SvEv	10	6	60	4/4
DBA-2/N	34	13	38	3/3

EmbryoMax Mouse and Rat Embryo Media & Reagents

Millipore offers the industry's widest selection of mouse embryo media and reagents. This includes M2, Modified M16, and the proprietary KSOM media formulations, which are manufactured using the highest quality raw materials available. In addition to liquid formats, these media formulations are also available in a dry powder format. Most importantly, our mouse embryo culture media and reagents (or their components) have been tested and approved for use on mouse embryos.

Mouse Embryo Media – Powder Kits

The embryo culture media in a dry powder format offers greatly increased shelf life, easy and quick preparation and the freshest nutrients to support the development of your embryos in culture. Just add the included sterile diluent to the powder, mix gently and filter to prepare fresh medium with the correct pH and osmolarity.

Two week old chimeric mice (left) generated from targeted B6-White ES cells injected into host C57BL/6 blastocysts. Germline transmission from the first litter was obtained.

Cryopreserved Mouse Embryos

The cryopreserved one-cell/pronuclear and eight-cell mouse embryos are capable of supporting transgenic procedures. Typical in vivo survival closely resembles that of freshly collected embryos. Using cryopreserved embryos greatly reduces the costs and concerns associated with live animals. In addition, cryopreserved embryos are always available and ready for your experimental demands and are not subject to seasonal variations in embryo yield. The customer specifies the mouse strain, stage of embryo, and the delivery schedule. Cryopreserved embryos are packaged as 25 embryos to a straw and shipped in dry nitrogen containers. The cryopreserved embryos are generated from animals housed in a SPF facility.

Mouse Embryonic Stem Cell Lines

The PluriStem™ range of murine embryonic stem (ES) cells exhibits high targeting efficiencies and features cell lines derived from mice of different genetic backgrounds. The generation of genetically-modified mice, created by homologous recombination in embryonic stem cell lines, has become a fundamental tool for analyzing gene contribution. The influence of genetic background on phenotype is an important consideration in the selection of a mouse model. The time required to achieve congenic status, which can be 2 to 3 years using traditional backcrossing methods, stimulated the use of pure inbred strains, such as Black 6 (B6) mice for targeting.

Millipore’s new [INSERT LINK]B6-White™[INSERT LINK] murine ES cell line is the first commercially available C57BL6/tyr c-2J albino line that allows for rapid coat-color determination of successful chimerism in the B6 mouse strain. When B6White murine ES cells are injected into B6 blastocysts, the resulting chimeric mice are easily identified by their coat color (a mix of black and white patches), while non-chimeric littermates are black. These cells allow for the efficient generation of gene-targeted mice in a pure B6 genetic background, thus providing more experimental flexibility.

Additionally, the EmbryoMax range of mitotically arrested Primary Mouse Embryo Fibroblasts (PMEF) provides researchers with a convenient solution for ES cell culture, negating the need for time consuming feeder cell isolation and preparation. PMEF feeder cells are supplied as frozen vials containing 5–6 x 10⁶cells per vial at passage 3 (2–3 population doublings per passage).

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