This figure is related to Fig. 1. a–f, Modification of human embryo culture medium. To test human embryo development, human embryos at 5–6 d.p.f. were cultured on low attachment plate without any 3D extracellular matrix up to 14 d.p.f. By sequentially culturing in IVC1 (6–8 d.p.f.) and IVC2 (8–14 d.p.f.) media, 6.67% (2 out of 30 embryos) of embryos could survive until 14 d.p.f. a, Schematics of improving culture medium. Sodium lactate, sodium pyruvate and ROCK inhibitor (Y27632) were added to the IVC1 and IVC2 media, referred to as mIVC1 (6–8 d.p.f.) and mIVC2 (8–14 d.p.f.), respectively. Culture in mIVC1 and mIVC2 enabled 25% (4 out of 16 embryos) of human blastocysts to develop up to 14 d.p.f. (c). b, Representative developing embryos based on morphological observation. n = 16 independent embryos from three independent experiments. c, Representative embryos with abnormal development. Abnormal embryos displayed growth arrest or had obvious cell death or fragmentations. n = 13 independent embryos from three independent experiments. d, Representative staining of abnormal embryos with CK7, GATA6 and OCT4 at 14 d.p.f. In all six examined embryos, consistent data were obtained. e, Staining of developing embryos (2 out of 3 embryos) with CK7, GATA6 and OCT4 at 14 d.p.f. f, Quantification of developmental rates of human embryos cultured in control medium (IVC1 and IVC2) and modified medium (mIVC1 and mVIC2). n = 30 and 16 biologically independent embryos, respectively. Developmental rates of human embryos at 11 and 13 d.p.f. were based on the two following requirements by morphology: obvious expansion over culture and absence of obvious cell death mass and fragmented phenotypes. At 14 d.p.f., we determined the embryo development ratio by staining CK7 (TrB), GATA6 (PrE,) and OCT4 (EPI). g–q, Representative human embryo development after culture under four different 3D conditions over development. The limited number of embryos only enabled us to compare embryo development under four conditions. As the implantation time window is 8–10 d.p.f., we embedded embryos with Matrigel at 9 d.p.f. Embryo development was verified on basis of morphological observation and staining of specific markers for OCT4, GATA6 and CK7. g–j, Top, schematics of in vitro 3D culture of human blastocysts under different culture conditions. Middle, representative images of human embryos during development. Bottom, representative stained images of cultured human embryos under different 3D conditions at 14 d.p.f. g, Human embryos at 5–6 d.p.f. were cultured on low attachment plate without Matrigel (W/O Matr) up to 14 d.p.f. The outermost TrBs showed signs of apoptosis (blue arrowheads), as determined by morphological observations and CK7 staining, which suggests that the condition was unsuitable for TrB development and survival required for attachment. In total, 2 of 20 embryos (three independent experiments) survived up to 14 d.p.f. and displayed normal EPI, PrE and TrB development. h, Human embryos were embedded in 25% Matrigel at 9 d.p.f. for continuous culture up to 14 d.p.f. n = 25 embryos from three independent experiments. The invasion and outgrowth of TrBs were observed and embryos became flat, which suggests a higher concentration of Matrigel is advantageous to differentiation and development of TrBs, as confirmed by CK7 staining. i, Human embryos were embedded in 10% Matrigel on the new well, which was pre-coated with 100% Matrigel (30 μl) (Matr+10%Matr), at 9 d.p.f. for continuous culture up to 14 d.p.f. Although embryos displayed considerable expansion over culture, staining with lineage markers showed that EPIs in most embryos were lost over development. Only 1 of 33 embryos from three independent experiments grew to 14 d.p.f and was accompanied by EPI, PrE and TrB development. The negative outcome may indicate that high concentrations of Matrigel can inhibit EPI development. In h and i, red arrowheads indicate TrBs invading into Matrigel. j, Human embryos were embedded in 10% Matrigel at 9 d.p.f. for continuous culture up to 14 d.p.f. Compared with the 25% Matrigel and Matr+10% Matr conditions, human embryos in 10% Matrigel increased in size at the thickness (Z axis) and showed better 3D spatial structures. In total, 4 of 17 embryos from three independent experiments grew to 14 d.p.f. with normal development. k, Quantification of the mean diameter of human embryos cultured under different 3D conditions during development by analysis of 5–14 embryos from three independent experiments. Data are mean ± s.d. l, Quantification of human developmental embryos during culture in different 3D conditions. Data were based on morphological observations only. Human developing embryos met the two following requirements: obvious expansion over culture; absence of obvious cell death mass or fragmented phenotypes. m, Quantification of developing embryos in different 3D conditions. n = 20 (W/O Matr), 25 (25% Matr), 33 (Matr+10% Matr) and 17 (10% Matr) blastocysts. Developing embryos met the following requirements: obvious expansion over culture; absence of obvious cell death mass or fragmented phenotypes; and development of EPIs, PrEs and TrBs and formation of amnion identified by OCT4, GATA6 and CK7 staining. Although embryos in the 25% Matr and Matr+10% Matr culture conditions have normal morphologies, some embryos lacked OCT4+ EPIs or GATA6+ PrEs and gave rise to a high proportion of TrBs, which suggest that high concentrations of Matrigel could inhibit EPI development and promote TrB proliferation. n–q, Three-dimensional construction of human embryos cultured in 10% Matrigel (3 out of 3 embryos from three independent experiments). n, A representative 3D reconstruction of EPIs. Inset shows OCT4 staining of one section from the same embryo. o, A representative 3D reconstruction of SYS and amnion including an embryonic disc and an amniotic cavity (see Supplementary Video 1). p, Three-dimensional reconstruction of TrBs (see Supplementary Video 2). q, Three-dimensional magnification of TrBs close to the amnion side. Scale bars, 100 μm (phase-contrast) or 50 μm (staining). Source Data