[1] Human Embryo Culture

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1 [1] human embryo culture 3 [1] Human Embryo Culture By AMPARO MERCADER, DIANA VALBUENA, and CARLOS SIMÓN Abstract Human embryonic stem cells (hescs) are derived from preimplantation embryos. Approximately 60% of human embryos are blocked during in vitro development. Although statistics are inconclusive, experience demonstrates that hescs are more effectively derived from high quality embryos. In this way, optimal human embryo culture conditions are a crucial aspect in any derivation laboratory. Embryos can be cultured solely with sequential media or cocultured on a monolayer of a given cell type. This chapter explores general aspects of human embryonic development, the concept of sequential culture and coculture, and specific protocols and procedures in which the authors are experienced, including the results obtained. Introduction Human embryonic stem cells (hescs) are derived from preimplantationstage embryos, a process that involves culturing embryos to the blastocyst stage (Thomson et al., 1998). HESCs have also been isolated from morula stage embryos (Strelchenko et al., 2004) and even from later stage blastocysts (7 8 days) (Stojkovic et al., 2004). Although hesc lines have been derived from embryos of poor quality (Mitalipova et al., 2003), it is clear that hescs are more efficiently derived from high quality embryos (Oh et al., 2005; Simon et al., 2005). To optimize embryo development in vitro, it is essential to adopt a global approach to the embryo culture system that takes into account the media, gas phase, type of medium overlay, culture vessel, incubation chamber, ambient air quality, and the embryologists themselves. The concept of an embryo culture system highlights the interactions that exist not only between the embryo and its physical surroundings but also with all the parameters present in the laboratory. Only by taking such a holistic approach we can optimize embryo development in vitro as the previous step for optimal hesc derivation. Initially, the zygote cleaves into two daughter cells, which subsequently divide to form the morula 4 days later. The transcription of the embryonic genome first occurs between the four and eight cell stages (Braude et al., METHODS IN ENZYMOLOGY, VOL /06 $35.00 Copyright 2006, Elsevier Inc. All rights reserved. DOI: /S (06)

2 4 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [1] 1988), which constitutes a critical moment. Compaction of the individual blastomeres follows, and finally develop to the blastocyst stage. Approximately 40 50% of embryos arrest during in vitro development. Today, human embryology laboratories are faced not only with a multitude of embryo culture media from which to choose but also with various possibilities of how to use defined media or coculture systems. Human Embryo Development In the laboratory, embryo development from oocyte retrieval to the blastocyst stage occurs as follows: Day 0: The human oocyte is retrieved from the follicle. Day 1: Fertilization day. Polar bodies and pronuclei are visualized. Only fertilized eggs with two polar bodies and two pronuclei are considered to be correctly fertilized. Day 2: First cleavage. The embryos generally have two to four cells. Embryos are evaluated for number of blastomeres (n), rate (%) and type (n) of fragmentation, symmetry (n), compaction (n), and multinucleation and are classified accordingly (Fig. 1A). Example: an embryo with four cells, 10% of fragmentation equally distributed throughout, with blastomeres of a similar size, without compaction and with one cell with two nuclei is classified as 4, 10, III, 2, 0, 1 2. Day 3: The embryos have six to eight cells and are evaluated as indicated previously (Fig. 1B). Day 4: Subsequent divisions form a cell embryo: the morula stage. Individual blastomeres become indistinguishable as they come into close contact with each other. This phenomenon is named compaction. On day 4, the type of morula is classified as either morula or compacted morula (Fig. 1C). Day 5: Spaces appear between the compacting cells, resulting in the formation of an external layer of cells, known as the trophoblast, and a group of centrally located cells, known as the inner cell mass (ICM). At this stage of development, the embryo is called a blastocyst (Fig. 1D). Day 6: The blastocoelic cavity enlarges and causes the embryo to grow and begin to hatch out from the zona pellucida (ZP). Blastocyst expansion thins the ZP because of a series of expansions and contractions. Blastocysts are classified morphologically as follows: Early blastocyst: when spaces appear between the compaction (Fig. 2A).

3 [1] human embryo culture 5 A B C D FIG. 1. Embryo development. (A) Two cell embryo. (B) Eight cell embryo. (C) Compacted morula. (D) Expanded blastocyst. Cavitated blastocyst: when the blastocoelic cavity is more than 50% of the total volume (Fig. 2B). Expanded blastocyst: when the blastocoelic cavity enlarges in size, the embryo and a monolayer, also known as the trophectoderm (TE), and an ICM can be differentiated (Fig. 2C). Hatching blastocyst: the embryo begins to hatch out of the ZP. Hatched blastocyst: the embryo is outside the zona pellucida (Fig. 2D). The different parts of the blastocysts ICM and TE can also be classified morphologically. Inner Cell Mass There are four types of ICM: A. Dense and compact with many cells (Fig. 3A). B. Several cells and not compact (Fig. 3B).

4 6 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [1] A B D C FIG. 2. Types of blastocyst. (A) Early. (B) Cavitated. (C) Expanded. (D) Hatched. C. Very few cells (Fig. 3C). D. Absence of a true ICM (pseudoblastocyst) (Fig. 3D). Trophectoderm There are four types: A. Complete, with a monolayer of cells; forming a cohesive epithelium (Fig. 3E). B. Incomplete, with a lineal zone (Fig. 3F).

5 [1] human embryo culture 7 A B C D E F G H FIG. 3. (A D) Types of inner cell mass. (E H) Types of trophectoderm.

6 8 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [1] C. With few large cells (Fig. 3G). D. With degenerated cells (Fig. 3H). Example: An expanded blastocyst with an ICM of very few cells and a trophectoderm with a lineal zone is classified as BE (C, B). Embryo development does not always follow an ideal pattern, sometimes becoming delayed or blocked because of low quality or accelerating inappropriately because of chromosomal abnormalities. Furthermore, morphology can vary considerably and is difficult to interpret at the expected developmental stage. Human Embryo Culture: General Considerations The dramatic changes in embryo physiology, nutrient requirements, and nutrient gradients in the female reproductive tract have led to the formulation of two culture media that are applied at different stages of human embryo development. This is the practice of sequential culture media. On the other hand, the concept of cells helping cells, extended throughout many areas of cell biology, has prompted embryologists to coculture human embryos in the presence of other types of cells (feeder cells), resulting in the development of the coculture system (Simon et al., 1999; Mercader et al., 2003). Sequential Culture Several detailed treatises have been written on the composition of embryo culture media, focusing particularly on four components: glucose, amino acids, ethylenediaminetetraacetic acid (EDTA), and macromolecules. Studies in mammals, including humans (Conaghan et al., 1993; Quinn, 1995), have demonstrated the importance of relatively high concentrations of pyruvate and lactate and a relatively low level of glucose in the early stages, whereas the opposite metabolic conditions have been shown to be required at the blastocyst stage. Amino acids contained in culture media enhance human embryo development up until the blastocyst stage (Devreker et al., 1998). In particular, a switch occurs in amino acid requirements during embryo development (Lane and Gardner, 1997). The beneficial effects of divalent cation EDTA in embryo culture media have been extensively reported (Mehta et al., 1990), although said benefits are confined to the cleavage stage (Gardner and Lane, 1996; Gardner et al., 2000a). A commonly used protein source in human IVF and embryo culture has been patient serum, which is added to the culture medium at a concentration of 5 20%. However, recombinant human serum albumin (HSA) is now available, eliminating the problems associated with transfusion and permitting the standardization of media formulation (Gardner et al., 2000b).

7 [1] human embryo culture 9 In general, the sequential culture is composed of two different media designed to meet metabolic requirements throughout embryo development. The first of these media is designed to support the development of the zygote to the eight cell stage, whereas the second aids development from the eight cell stage to the blastocyst stage. Coculture System Even though the formulations of embryo culture media have improved significantly over the years and have, for the most part, a more physiological composition, embryo development in vitro still lags behind that in vivo. For this reason, the sequential system has been opened up to include the coculture strategy. The suggested beneficial effects of coculture include the secretion of embryotrophic factors such as nutrients and substrates, growth factors and cytokines (for a review, see Bavister [1995]), and the elimination of potentially harmful substances such as heavy metals and ammonium and free radical formation, thereby detoxifying the culture medium. Multiple cell types have been used for this purpose, including human reproductive tissues such as oviducts (Bongso et al., 1992, 1989; Ouhibi et al., 1989; Walker et al., 1997; Weichselbaum et al., 2002; Yeung et al., 1996), human endometrium (Barmat et al., 1998; De los Santos et al., 2003; Desai et al., 1994; Jayo t et al., 1995; Liu et al., 1999 ; Simó n et al., 1999; Spando rfer et al., 2002), oviduct endometrial sequential coculture (Bongso et al., 1994), cumulus cells (Carrell et al., 1999; Quinn and Margalit, 1996; Saito et al., 1994), granulosa cells (Fabbri et al., 2000; Plachot et al., 1993), non human cells (Wiemer et al., 1993), non human cell lines (D Estaing et al., 2001; Hu et al., 1997, 1998; Magl i et al., 1995; Menezo et al., 1990, 1992 ; Sakk as et al., 1994; Schillaci et al., 1994; Turner and Lent on, 1996; Van Blerkom, 1993; Veiga et al., 1999), and even cells from ovarian carcino ma ( Ben Chet rit et al., 1996). As a consequence, the reported effects of this technology on embryonic development are cell, tissue, and species nonspecific. We have developed a coculture system using autologous human endometrial epithelial cells (Simon et al., 1999), and we routinely use this system as a clinical program in our center (Mercader et al., 2003). Protocols Protocol of Sequential Culture 1. On day 0 (oocyte retrieval day), culture dishes with human tubal fluid (HTF) (IVI, Barcelona, Spain) culture drops of 50 l, overlaid with oil, are incubated overnight in a 5% CO 2 incubator.

8 10 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [1] 2. On day 1, embryo fertilization is assessed. All correctly fertilized oocytes are rinsed with the HTF in the culture dish before being transferred to the culture drops. 3. On day 2, embryos are assessed to identify whether they have reached the cleavage stage. Culture drops containing 50 l of CCM medium (Vitrolife AB, Kungsbacka, Sweden) are placed in a culture dish and overlaid with oil. The culture dish is placed in the incubator overnight. 4. On day 3, embryo cleavage is assessed. Embryos are transferred from the HTF medium into the CCM medium, where they remain until derivation (day 5 or 6). 5. On day 4, the embryos are maintained in CCM. Culture drops containing 50 l of CCM medium are placed in a culture dish and overlaid with oil. The culture dish is placed in the incubator overnight. 6. On days 5 and 6, embryos are assessed according to the morphological classification previously indicated. Derivation is performed when good quality blastocysts are achieved at day 5 or 6. Protocols for the Embryo Coculture System with Human Endometrial Epithelial Cells (Simon et al., 1999; Mercader et al., 2003) Reagents for Endometrial Culture COLLAGENASE TYPE IA. The digestion is performed with 0.1% collagenase in DMEM. 1. Add 10 ml of water for embryo transfer to 100 mg of collagenase. 2. Stock concentration is 10 mg/ml. 3. Digestion volume is 10 ml; therefore, add 0.1 ml collagenase stock (10) to 0.9 ml DMEM (10) to obtain 0.1 % collagenase in DMEM. 4. Store in 1 ml aliquots at 20. DULBECCO s MODIFIED EAGLE s MEDIUM (DMEM). This is a liquid media, and no supplements are added. MCDB 105. This is a powdered medium. Prepare with water for embryo transfer as described in the following. Store in the dark at Measure 90% of the final required volume of embryo transfer water. 2. Add the powdered medium. Stir until dissolved, but do not heat. The medium is yellow in color. 3. Rinse original package with a small amount of water to remove all traces of powder. Add to solution.

9 [1] human embryo culture Adjust the ph of the medium. The ph at room temperature must be Although the final ph should be 7.4, we adjust it to 7.2 because at 37, ph increases units. Add 4 5 ml of 1 M NaOH and check ph. Add 1 M NaOH until ph ¼ Add additional water to bring the solution to final volume. 6. Sterilize immediately by filtration using a membrane with a porosity of 0.22 m. FUNGIZONE. The vial contains 250 g/ml of Amphotericin B. 1. Rehydrate with 20 ml of embryo transfer water. The recommended final concentration is between 0.25 and 2.5 g/ml. Our working dilution is 0.5 g/ml; therefore, add 2 l/ml to the medium. GENTAMICIN. The vial contains 50 mg/ml. Our working concentration is 100 g/ml; therefore, add 2 l/ml to the medium. INSULIN. Insulin promotes the uptake of glucose and amino acids and has a mitogenic effect. It is stable at 2 8 for 1 year. Soluble insulin is available in acidified water. Our working dilution is 5 g/ml. 1. For a vial of 100 mg, to prepare a 10 mg/ml stock solution, add 10 ml of acidified water (ph 2.0) (add 100 l of glacial acetic acid). 2. Add 0.5 l/ml to the medium to obtain the correct working concentration. HUMAN SERUM ALBUMIN. This serum is used to promote cell attachment. Appropriate aliquots (volume recommended is 40 ml) should be prepared using sterile containers. The serum should be stored at 20. Preparation of Endometrial Epithelial Cell Medium We use two basic media (DMEM and MCDB 105), supplemented with 10% HSA and insulin. In addition, antibiotics and antimycotics are added to control possible contamination. The medium is composed of: 3 DMEM:1 MCDB 105 supplemented with 10% HSA, gentamicin (100 g/ml), Fungizone (0.5 g/ml), and insulin (5 g/ml). It is sterilized by passing it through a 0.22 m filter and is then stored in aliquots at 4. ENDOMETRIAL CULTURE. Endometrial biopsy specimens are obtained in the luteal phase with a catheter (Gynetics, Amsterdam, The Netherlands).

10 12 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [1] Epithelial and stromal endometrial cells are isolated as described in the following: 1. Mince the biopsy specimens into small pieces of less than 1 mm in length. 2. The minced biopsy pieces are placed in a conical tube with 10 ml of 0.1% collagenase type IA. 3. The biopsy is exposed to mild collagenase digestion through agitation for 1 h in a 37 water bath. 4. Stand the tube in a vertical position for 10 min in a horizontal laminar flow. 5. Remove the supernatant (with stromal cells) and wash by resuspending the pellet (glandular and epithelial cells) three times, for 5 min each time, in 3 5 ml of DMEM. 6. Finally, resuspend the pellet in 4 5 ml of 1% HSA in DMEM. Recover the mixture into a culture flask (Falcon, Becton Dickinson, NJ). Incubate the flask for 15 min. 7. Recover the supernatant into a fresh flask and add 3 ml of 1% HSA in DMEM. Incubate this second flask for 15 min. 8. Recover all the supernatant and place it in a tube. Check the volume. 9. Prepare l of endometrial epithelial cell medium and add l of recovered supernatant with cells into culture wells. 10. Glandular epithelial cells are cultured for approximately 4 6 days until confluent (monolayer) (Fig. 4). The monolayer of endometrial epithelial cells is used for embryo coculture. Protocol for Embryo Coculture on Human Endometrial Epithelial Cells On day 0 (oocyte retrieval day), culture dishes with IVF medium (Vitrolife AB, Kungsbacka, Sweden) are placed in the incubator. Culture drops of 50 l are placed in a culture dish and overlaid with oil. The medium is incubated overnight in 5% CO 2. On day 1, embryo fertilization is assessed. All correctly fertilized oocytes are rinsed in the drops in the culture dish before being transferred to the IVF culture drops. IVF and CCM media are left in the incubator (0.5 ml of each one/zygote) overnight. On day 2, embryos are assessed to detect whether they have reached the cleavage stage. A single embryo is cocultured with 1 ml of IVF:CCM (1:1) on an endometrial epithelial monolayer in individual wells on a 24 multiwell tissue culture plate (Falcon, Becton Dickinson).

11 [1] human embryo culture 13 A B C1 C2 FIG. 4. Endometrial epithelial cells culture. (A) On day 0. (B) Proliferation. (C) Monolayer. On days 4 6. On day 3, embryo development is assessed. Embryos are transferred to CCM drops, and the culture dish is placed for min in the incubator. The embryos are then assessed. The IVF:CCM medium is removed, and 1 ml of CCM medium is placed in each well with one embryo.

12 14 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [1] On day 4, embryo development is assessed. On days 5 and 6, embryos are morphologically classified. Results Human embryonic development using the two previously described culture systems was compared in a total of 3508 embryos from 474 patients attending the Instituto Valenciano de Infertilidad between January and December Patients were treated for infertility and divided into two groups according to whether they use their own oocytes (IVF) or receive ova from donors (ovum donation program). In addition, each group was divided into two subgroups in accordance with the culture system used: coculture versus sequential culture. We have analyzed, at different stages of development, the blastocyst rates achieved with sequential culture versus our coculture system with endometrial epithelial cells. A chi square probability test was used to analyze the blastocyst rates of each group. P values < 0.05 were considered to be statistically significant. In patients with IVF, statistical differences were observed in the rates of expanded blastocysts (p < 0.05) obtained and in the total blastocyst rates in coculture versus sequential media (56.2 vs. 47.3% [p < 0.01], respectively) (Table I). In patients with ovum donation (Table II), a statistical increase in cavitated and hatching blastocysts (p < 0.05) was obtained with the coculture system, whereas the total blastocyst rate augmented differently, depending on the culture system used: 65.2% (coculture) vs. 51.9% (sequential) (p < ). In conclusion, the experience acquired in the IVF laboratory over the past decades allows us to optimize the culture system for human embryos and therefore improve both implantation and derivation success rates. Many embryos used for hesc derivation have been frozen and must TABLE I COMPARATIVE EMBRYO DEVELOPMENT IN PATIENTS UNDERGOING IN VITRO FERTILIZATION Type of blastocyst Coculture system (%) Sequential (%) p Early 31 (9.4) 70 (8.1) ns Cavitated 36 (10.9) 96 (11.1) ns Expanded 83 (25.1) 161 (18.5) < 0.05 Hatching 35 (10.5) 76 (8.7) ns Hatched 1 (0.3) 8 (0.9) ns Total 186 (56.2) 411 (47.3) < 0.01

13 [1] human embryo culture 15 TABLE II COMPARATIVE EMBRYO DEVELOPMENT IN PATIENTS UNDERGOING OOCYTE DONATION Type of blastocyst Coculture system (%) Sequential (%) p Early 57 (7.3) 104 (6.8) ns Cavitated 128 (16.5) 160 (10.4) < Expanded 207 (26.6) 355 (23.2) ns Hatching 104 (13.4) 159 (10.4) < 0.05 Hatched 11 (1.4) 18 (1.1) ns Total 507 (65.2) 796 (51.9) < therefore be thawed following a specific protocol, after which the indicated culture systems can be applied. Acknowledgments We express our gratitude to Miodrag Stojkovic for critical proofreading of this manuscript. References Barmat, L. I., Liu, H. C., Spandorfer, S. D., Xu, K., Veeck, L., Damario, M. A., and Rosenwaks, Z. (1998). Human preembryo development on autologous endometrial coculture versus conventional medium. Fertil. Steril. 70, Bavister, B. D. (1995). Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update 1, Ben Chetrit, A., Jurisicova, A., and Casper, R. F. (1996). Coculture with ovarian cancer cell enhances human blastocyst formation in vitro. Fertil. Steril. 65, Bongso, A., Fong, C. Y., Ng, S. C., and Ratnam, S. (1994). Human embryonic behaviour in a sequential human oviduct endometrial coculture system. Fertil. Steril. 61, Bongso, A., Ng, S. C., Fong, C. Y., Anandakumar, C., Marshall, B., Edirisinghe, R., and Ratnam, S. (1992). Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture. Fertil. Steril. 58, Bongso, A., Soon Chye, N., Sathananthan, H., Lian, N. P., Rauff, M., and Ratnam, S. (1989). Improved quality of human embryos when co cultured with human ampullary cells. Hum. Reprod. 4, Braude, P., Bolton, V., and Moore, S. (1988). Human gene expression first occurs between the four and eight cell stages of preimplantation development. Nature 332, Carrell, D. T., Peterson, C. M., Jones, K. P., Hatasaka, H. H., Udoff, L. C., Cornwell, C. E., Thorp, C., Kuneck, P., Erickson, L., and Campbell, B. (1999). A simplified coculture system using homologous, attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro fertilization. J. Assist. Reprod. Genet. 16, Conaghan, J., Handyside, A. H., Winston, R. M., and Leese, H. J. (1993). Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J. Reprod. Fertil. 99,

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16 18 IN VITRO EXPERIMENTATION AND RESEARCH TOOLS [2] Veiga, A., Torrello, M. J., Menezo, Y., Busquets, A., Sarrias, O., Coroleu, B., and Barri, P. N. (1999). Use of co culture of human embryos on Vero cells to improve clinical implantation rate. Hum. Reprod. 14, Walker, D. J., Vlad, M. T., and Kennedy, C. R. (1997). Establishment of human tubal epithelial cells for coculture in an IVF program. J. Assist. Reprod. Genet. 14, Weichselbaum, A., Paltieli, Y., Philosoph, R., Rivnay, B., Coleman, R., Seibel, M. M., and Bar Ami, S. (2002). Improved development of very poor quality human preembryos by coculture with human fallopian ampullary cells. J. Assist. Reprod. Genet. 19, Wiemer, K. E., Hoffman, D. I., Maxson, W. S., Eager, S, Muhlberger, B., Fiore, I., and Cuervo, M. (1993). Embryonic morphology and rate of implantation of human embryos following co culture on bovine oviductal epithelial cells. Hum. Reprod. 8, Yeung, W. S., Lau, E. Y., Chan, S. T., and Ho, P. C. (1996). Coculture with homologous oviductal cells improved the implantation of human embryos a prospective randomized control trial. J. Assist. Reprod.Genet. 13, [2] Characterization and Evaluation of Human Embryonic Stem Cells By CHUNHUI XU Abstract Human embryonic stem cells (hescs) provide great opportunities for regenerative medicine, pharmacological and toxicological investigation, and the study of human embryonic development. These applications require proper derivation, maintenance, and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hescs possess several unique features, including their extensive proliferation capacity in the undifferentiated state, ability to maintain a normal karyotype after long term culture, expression of markers characteristic of stem cells, high constitutive telomerase activity, and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hescs. Introduction hescs were first successfully isolated from inner cell masses (ICMs) of preim plantati on embry os or blastocys ts ( Thom son et al., 1998 ). ICM s were obtained by removal of trophoblast, the outer layer of blastocysts, through immunosurgery, and the cells were plated onto a layer of mitotically inactivated mouse embryonic fibroblast (MEF) feeders to prevent cells METHODS IN ENZYMOLOGY, VOL /06 $35.00 Copyright 2006, Elsevier Inc. All rights reserved. DOI: /S (06)

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